Relaxin immunoglobulin fusion proteins and methods of use

ABSTRACT

Disclosed herein are relaxin immunoglobulin fusion proteins useful for the treatment or prevention of a disease or condition in a subject.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/060,384, filed Jun. 7, 2018, which is a U.S. National Stage entry ofInternational Application No. PCT/US2016/065779, filed Dec. 9, 2016;which claims the benefit of U.S. Provisional Application No. 62/265,344,filed Dec. 9, 2015, each of which are incorporated by reference hereinin their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 26, 2021, isnamed 41135-756-301-SEQ.txt and is 92.4 KB in size.

BACKGROUND OF THE INVENTION

Relaxin is expressed mainly in the corpus luteum, in both pregnant andnon-pregnant females, rising to a peak within approximately 14 days ofovulation, and then declining in the absence of pregnancy, resulting inmenstruation. During the first trimester of pregnancy, levels rise andadditional relaxin is produced by the decidua. Relaxin expression peaksduring the 14 weeks of the first trimester and at delivery. It is knownto mediate the hemodynamic changes that occur during pregnancy, such asincreased cardiac output, increased renal blood flow, and increasedarterial compliance. It also relaxes other pelvic ligaments and softensthe pubic symphysis. In males, relaxin enhances motility of sperm insemen.

Outside the reproductive system, relaxin affects collagen metabolism,inhibiting collagen synthesis and enhancing its breakdown by increasingmatrix metalloproteinases. Relaxin also enhances angiogenesis and is apotent renal vasodilator.

Relaxin interacts with the relaxin receptor LGR7 (RXFP1) and LGR8(RXFP2), which belong to the G protein-coupled receptor superfamily.Relaxin receptors have been found in the heart, smooth muscle, theconnective tissue, and central and autonomous nervous system.

SUMMARY OF THE INVENTION

Disclosed herein are relaxin immunoglobulin fusion proteins and methodsof using the same for the treatment of various diseases and conditions.The disease or condition may be acute, for example, acute heart failure,acute coronary syndrome with cardiac dysfunction, ischemia reperfusionassociated with solid organ transplantation, cardiopulmonary bypass,ischemic stroke, or preeclampsia. The disease or condition may bechronic, for example, diffuse scleroderma, chronic heart failure,diabetic nephropathy, cirrhosis, portal hypertension, atrialfibrillation, cardiac fibrosis, and diabetic wound healing. The methodsand compositions may also be used to improve the delivery of a relaxinpeptide to target cells, tissues, or tumors.

In one aspect, provided herein are compositions comprising: (a) relaxintherapeutic peptide comprising a relaxin B chain connected via a peptidelinker to a relaxin A chain; and (b) an antibody variable domaincomprising SEQ ID NO: 75: SMITX(1)X(2)X(3)FDV, wherein X(1) is selectedfrom F, A, G, and P; X(2) is selected from G, A, S, T, and P; and X(3)is selected from G, A, V, L, and P; and wherein the relaxin therapeuticpeptide is connected to the amino-terminus of the antibody variabledomain with a connecting peptide. In some embodiments, the antibodyvariable domain is modified from a heavy chain variable domain of apalivizumab antibody comprising SEQ ID NO: 74, and the compositionexhibits reduced binding to RSV-F as compared to the palivizumabantibody. In some embodiments, X(1) is F. In some embodiments, X(1) isA. In some embodiments, X(1) is G. In some embodiments, X(1) is P. Insome embodiments, X(2) is G. In some embodiments, X(2) is A. In someembodiments, X(2) is S. In some embodiments, X(2) is T. In someembodiments, X(2) is P. In some embodiments, X(3) is G. In someembodiments, X(3) is A. In some embodiments, X(3) is V. In someembodiments, X(3) is L. In some embodiments, X(3) is P. In someembodiments, the antibody variable domain further comprises one or moreof SEQ ID NOS: 72 and 73. In some embodiments, the composition furthercomprises an antibody sequence comprising one or more of SEQ ID NOS: 89or 90. In some embodiments, the composition further comprises anantibody sequence comprising SEQ ID NO: 92 (FQX(4)X(5)GYPFT), whereinX(4) is selected from G, Y, F, W, P, L, V, and A; and X(5) is selectedfrom S, N, G, A, V, L, and P. In some embodiments X(4) is G. In someembodiments X(4) is Y. In some embodiments X(4) is F. In someembodiments X(4) is W. In some embodiments X(4) is P. In someembodiments X(4) is L. In some embodiments X(4) is V. In someembodiments X(4) is A. In some embodiments, X(5) is S. In someembodiments, X(5) is N. In some embodiments, X(5) is G. In someembodiments, X(5) is A. In some embodiments, X(5) is V. In someembodiments, X(5) is L. In some embodiments, X(5) is P. In someembodiments, X(4) is Y and X(5) is S. In some embodiments, X(4) is G andX(5) is N. In some embodiments, the relaxin B chain comprises an aminoacid sequence of SEQ ID NO: 46. In some embodiments, the relaxin A chaincomprises an amino acid sequence of SEQ ID NO: 47. In some embodiments,the peptide linker comprises at least about 4 consecutive amino acidscomprising any combination of G and S amino acids. In some embodiments,the connecting peptide comprises at least about 4 consecutive aminoacids comprising any combination of G and S amino acids. In someembodiments, the composition further comprises an Fc region of anantibody and the Fc region comprises one or more mutations to reduceantibody-dependent cellular cytotoxicity. In some embodiments, the Fcregion comprises an amino acid sequence at least about 90% identical toSEQ ID NO: 98.

In one aspect, provided herein are compositions comprising: (a) relaxintherapeutic peptide comprising a relaxin B chain connected via a peptidelinker to a relaxin A chain; and (b) an antibody variable domaincomprising a modified heavy chain variable domain of a palivizumabantibody comprising SEQ ID NO: 74 having reduced binding to RSV-F ascompared to the palivizumab antibody; wherein the relaxin therapeuticpeptide is connected to the amino-terminus of the antibody variabledomain with a connecting peptide. In some embodiments, the antibodyvariable domain comprises SEQ ID NO: 75: SMITX(1)X(2)X(3)FDV, whereinX(1) is selected from F, A, G, and P; X(2) is selected from G, A, S, T,and P; and X(3) is selected from G, A, V, L, and P. In some embodiments,X(1) is F. In some embodiments, X(1) is A. In some embodiments, X(1) isG. In some embodiments, X(1) is P. In some embodiments, X(2) is G. Insome embodiments, X(2) is A. In some embodiments, X(2) is S. In someembodiments, X(2) is T. In some embodiments, X(2) is P. In someembodiments, X(3) is G. In some embodiments, X(3) is A. In someembodiments, X(3) is V. In some embodiments, X(3) is L. In someembodiments, X(3) is P. In some embodiments, the antibody variabledomain further comprises one or more of SEQ ID NOS: 72 and 73. In someembodiments, the composition further comprises an antibody sequencecomprising one or more of SEQ ID NOS: 89 or 90. In some embodiments, thecomposition further comprises an antibody sequence comprising SEQ ID NO:92 (FQX(4)X(5)GYPFT), wherein X(4) is selected from G, Y, F, W, P, L, V,and A; and X(5) is selected from S, N, G, A, V, L, and P. In someembodiments, X(4) is Y and X(5) is S. In some embodiments X(4) is Y. Insome embodiments X(4) is F. In some embodiments X(4) is W. In someembodiments X(4) is P. In some embodiments X(4) is L. In someembodiments X(4) is V. In some embodiments X(4) is A. In someembodiments, X(5) is S. In some embodiments, X(5) is N. In someembodiments, X(5) is G. In some embodiments, X(5) is A. In someembodiments, X(5) is V. In some embodiments, X(5) is L. In someembodiments, X(5) is P. In some embodiments, X(4) is G and X(5) is N. Insome embodiments, the relaxin B chain comprises an amino acid sequenceof SEQ ID NO: 46. In some embodiments, the relaxin A chain comprises anamino acid sequence of SEQ ID NO: 47. In some embodiments, the peptidelinker comprises at least about 4 consecutive amino acids comprising anycombination of G and S amino acids. In some embodiments, the connectingpeptide comprises at least about 4 consecutive amino acids comprisingany combination of G and S amino acids. In some embodiments, thecomposition further comprises an Fc region of an antibody and the Fcregion comprises one or more mutations to reduce antibody-dependentcellular cytotoxicity. In some embodiments, the Fc region comprises anamino acid sequence at least about 90% identical to SEQ ID NO: 98.

In another aspect, provided herein are methods of treating a disease orcondition in an individual in need thereof. In some embodiments, themethod comprises administering to the individual a compositioncomprising a relaxin therapeutic peptide; wherein the composition isintravenously or subcutaneously administered in less than or equal to 6hours. In some embodiments, the composition is administered in less thanor equal to 4, 3, 2 or 1 hours. In some embodiments, the composition isadministered during a treatment period of about one day to about 1 week.In some embodiments, the composition is administered about 1, 2, 3, 4,5, 6 or 7 times. In some embodiments, the disease or condition isselected from acute heart failure, acute coronary syndrome with cardiacdysfunction, ischemia reperfusion associated with solid organtransplantation, cardiopulmonary bypass, ischemic stroke, orpreeclampsia. In some embodiments, the disease or condition is solidorgan transplantation and the organ is selected from the lung, kidney,liver and heart. In some embodiments, the composition is administeredduring a treatment period lasting at least about one week. In someembodiments, the composition is administered twice per week, once perweek, four times per month, three times per month, twice per month, onceper month, or once per every two months. In some embodiments, thedisease or condition is selected from diffuse scleroderma, chronic heartfailure, diabetic nephropathy, cirrhosis, portal hypertension, atrialfibrillation, cardiac fibrosis, and diabetic wound healing.

In another aspect, provided herein are methods of treating a disease orcondition in an individual in need thereof. In some embodiments, themethod comprises administering to the individual a compositioncomprising a relaxin therapeutic peptide; wherein the composition isadministered to the eye. In some embodiments, the composition isadministered in a liquid solution or suspension. In some embodiments,the composition comprises a relaxin therapeutic peptide connected to anamino-terminus of an antibody variable domain via a connecting peptide.In some embodiments, the relaxin polypeptide comprises a relaxin B chainand a relaxin A chain connected by a peptide linker. In someembodiments, the relaxin polypeptide comprises (a) relaxin therapeuticpeptide comprising a relaxin B chain connected via a peptide linker to arelaxin A chain; and (b) an antibody variable domain comprising SEQ IDNO: 75: SMITX(1)X(2)X(3)FDV, wherein X(1) is selected from F, A, G, andP; X(2) is selected from G, A, S, T, and P; and X(3) is selected from G,A, V, L, and P; and wherein the relaxin therapeutic peptide is connectedto the amino-terminus of the antibody variable domain with a connectingpeptide. In some embodiments, the antibody variable domain is modifiedfrom a heavy chain variable domain of a palivizumab antibody comprisingSEQ ID NO: 74, and the composition exhibits reduced binding to RSV-F ascompared to the palivizumab antibody. In some embodiments, X(1) is F. Insome embodiments, X(1) is A. In some embodiments, X(1) is G. In someembodiments, X(1) is P. In some embodiments, X(2) is G. In someembodiments, X(2) is A. In some embodiments, X(2) is S. In someembodiments, X(2) is T. In some embodiments, X(2) is P. In someembodiments, X(3) is G. In some embodiments, X(3) is A. In someembodiments, X(3) is V. In some embodiments, X(3) is L. In someembodiments, X(3) is P. In some embodiments, X(2) is G. In someembodiments, X(2) is A. In some embodiments, X(2) is G. In someembodiments, the antibody variable domain further comprises one or moreof SEQ ID NOS: 72 and 73. In some embodiments, the relaxin polypeptidefurther comprises an antibody sequence comprising one or more of SEQ IDNOS: 89 or 90. In some embodiments, the relaxin polypeptide furthercomprises an antibody sequence comprising SEQ ID NO: 92(FQX(4)X(5)GYPFT), wherein X(4) is selected from G, Y, F, W, P, L, V,and A; and X(5) is selected from S, N, G, A, V, L, and P. In someembodiments X(4) is Y. In some embodiments X(4) is F. In someembodiments X(4) is W. In some embodiments X(4) is P. In someembodiments X(4) is L. In some embodiments X(4) is V. In someembodiments X(4) is A. In some embodiments, X(5) is S. In someembodiments, X(5) is N. In some embodiments, X(5) is G. In someembodiments, X(5) is A. In some embodiments, X(5) is V. In someembodiments, X(5) is L. In some embodiments, X(5) is P. In someembodiments, In some embodiments, X(4) is Y and X(5) is S. In someembodiments, X(4) is G and X(5) is N. In some embodiments, the relaxin Bchain comprises an amino acid sequence of SEQ ID NO: 46. In someembodiments, the relaxin A chain comprises an amino acid sequence of SEQID NO: 47. In some embodiments, the peptide linker comprises at leastabout 4 consecutive amino acids comprising any combination of G and Samino acids. In some embodiments, the connecting peptide comprises atleast about 4 consecutive amino acids comprising any combination of Gand S amino acids. In some embodiments, the relaxin polypeptide furthercomprises an Fc region of an antibody and the Fc region comprises one ormore mutations to reduce antibody-dependent cellular cytotoxicity. Insome embodiments, the Fc region comprises an amino acid sequence atleast about 90% identical to SEQ ID NO: 98.

Further provided herein are methods of treating cardiovascular diseasein an individual in need thereof, comprising administering an effectiveamount of a relaxin immunoglobulin fusion protein described herein toreduce at least one symptom of the cardiovascular disease. In someembodiments, the cardiovascular disease is selected from one or more ofthe group consisting of acute heart failure, congestive heart failure,compensated heart failure, decompensated heart failure, acutedecompensated heart failure, right heart failure, left heart failure,global failure, ischemic cardiomyopathy, dilated cardiomyopathy, heartfailure associated with congenital heart defects, heart failureassociated with heart valve defects, mitral stenosis, mitralinsufficiency, aortic stenosis, aortic insufficiency, tricuspidstenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary valveinsufficiency, heart failure associated with combined heart valvedefects, myocardial inflammation (myocarditis), chronic myocarditis,acute myocarditis, viral myocarditis, diabetic heart failure, alcoholiccardiomyopathy, heart failure associated with cardiac storage disorders,diastolic heart failure, and systolic heart failure, fibrosis of theheart, thromboembolic disorders, reperfusion damage following ischemia,micro- and macrovascular lesions (vasculitis), arterial and venousthromboses, edemas, ischemias, myocardial infarction, stroke, transientischemic attack, cardio protection in connection with coronary arterybypass operations, cardio protection in connection with primarypercutaneous transluminal coronary angioplasties (PTCAs), PTCAs afterthrombolysis, rescue PTCA, heart transplants and open-heart operations,organ protection in connection with transplants, bypass operations,catheter examinations and other surgical procedures, metabolic syndrome,dyslipemia, diastolic dysfunction, familial hypercholesterolemia,isolated systolic hypertension, primary hypertension, secondaryhypertension, left ventricular hypertrophy, arterial stiffnessassociated with long-term tobacco smoking, arterial stiffness associatedwith obesity, arterial stiffness associated with age, andhypercholesterolemia. In some embodiments, cardiovascular disease isheart failure. In some embodiments, the symptom is selected from one ormore of the group consisting of chest pain, shortness of breath, pain,numbness, weakness, rapid pulse, slow pulse, lightheadedness, dizziness,swelling of the limbs, and fainting.

Also provided herein are methods of treating fibrosis in an individualin need thereof, comprising administering an effective amount of arelaxin immunoglobulin fusion protein provided herein sufficient toreduce at least one symptom of the fibrosis. In some embodiments, thefibrosis is selected from one or more of the group consisting ofpulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis,cirrhosis, endomyocardial fibrosis, myocardial infarction, atrialfibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis,progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn'sdisease, keloid, scleroderma/systemic sclerosis, arthrofibrosis,Peyronie's disease, Dupuytren's contracture, and adhesive capsulitis.Also provided herein are methods of treating acute heart failure, acutecoronary syndrome with cardiac dysfunction, ischemia reperfusionassociated with solid organ transplantation, cardiopulmonary bypass,ischemic stroke, preeclampsia, diffuse scleroderma, chronic heartfailure, diabetic nephropathy, cirrhosis, portal hypertension, atrialfibrillation, cardiac fibrosis, diabetic wound healing, or a combinationthereof, comprising administering an effective amount of a relaxinimmunoglobulin fusion protein provided herein.

Also provided herein, are genetic constructs comprising a polynucleotideencoding a relaxin immunoglobulin fusion protein provided herein.Further provided herein are expression vector comprising the geneticconstructs disclosed herein. Further provided herein are mammalianexpression hosts comprising the expression vectors disclosed herein.Further provided herein are methods of producing a relaxinimmunoglobulin fusion protein comprising: transfecting the expressionvector of claim 45 into a mammalian cell culture; growing the cellculture in an expression medium at a controlled temperature andpercentage CO2; and harvesting the secreted immunoglobulin fusionprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe disclosure, will be better understood when read in conjunction withthe appended figures. It should be understood, however, that thedisclosure is not limited to the precise examples shown. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1A, FIG. 1B, and FIG. 1C show SDS-PAGE gels of purifiedpalivizumab-relaxin fusion proteins.

FIG. 2A, FIG. 2B, and FIG. 2C depict graphs of the activities ofpalivizumab-relaxin fusion proteins.

FIG. 3A and FIG. 3B depict graphs of palivizumab-relaxin fusionprotein/RSV-epitope binding assay.

FIG. 4 shows binding of palivizumab-relaxin fusion proteins to RSV.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F show SDS-PAGEgels of purified palivizumab-relaxin fusion proteins.

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, and FIG. 6F show graphs ofthe activities of palivizumab-relaxin fusion proteins.

FIG. 7A and FIG. 7B show graphs of the activities of palivizumab-relaxinfusion proteins.

FIG. 8A and FIG. 8B show protein characterization of palivizumab-relaxinfusion protein RLX303.

FIG. 9A, FIG. 9B, and FIG. 9C show serum stability of relaxin-2 peptideand palivizumab-relaxin fusion proteins RLX302 and RLX303.

FIG. 10 shows mouse pharmacokinetic analysis of palivizumab-relaxinfusion proteins.

FIG. 11A and FIG. 11B show rat pharmacokinetic analysis ofpalivizumab-relaxin fusion proteins.

FIG. 12A and FIG. 12B show pharmacodynamics analysis ofpalivizumab-relaxin fusion proteins.

DETAILED DESCRIPTION OF THE INVENTION

Relaxin amino-terminal palivizumab fusions with reduced binding torespiratory syncytial virus (RSV) proteins which have a relaxin peptidefused to the amino-terminus of a palivizumab heavy chain polypeptide areuseful in treating diseases responsive to relaxin peptide. Reducingbinding to RSV proteins is achieved by mutating amino acid residues inthe CDR portions of the palivizumab amino acid sequence. Furthertherapeutic value is obtained by reducing ADCC elicited by thepalivizumab by mutating amino acid residues in the Fc portion of thepalivizumab amino acid sequence.

Disclosed herein are amino-terminal immunoglobulin fusion proteinscomprising a relaxin peptide, and methods of use for the treatment of adisease or condition responsive to a relaxin peptide. According to onefeature of the subject matter described herein, an amino-terminalimmunoglobulin fusion protein comprises (a) a first immunoglobulinregion; and (b) a relaxin peptide connected to the amino terminus of thefirst immunoglobulin region with a connecting peptide. The firstimmunoglobulin region may be part of an immunoglobulin heavy chain, andthe immunoglobulin fusion protein further comprises a secondimmunoglobulin region of an immunoglobulin light chain. As used herein,an immunoglobulin region may include a variable domain such that therelaxin peptide is connected to the amino-terminus of a variable domainof the immunoglobulin region. The first and/or second immunoglobulinregion may comprise part of an antibody variable domain that does notform a binding site specific for a human antigen. In some cases, theantibody variable domain is a palivizumab variable domain, or a variabledomain modified from a palivizumab variable domain. The variable domainmay be modified from palivizumab to attenuate or eliminate RSV binding.

Exemplary amino-terminal immunoglobulin fusion proteins are depicted inFormulas I-XXXII, wherein T is a therapeutic peptide or a portion of atherapeutic peptide, C is a connecting peptide, A is an immunoglobulinregion, P is a protease site, L is a linker, and I is an internallinker. The therapeutic peptide may be a relaxin peptide comprising oneor a combination of relaxin B chain, relaxin A chain, and C-peptide orlinker peptide.

Formula Immunoglobulin fusion protein I T¹-A¹ II T¹-C-A¹ III T¹-C-P¹-A¹IV T¹-P¹-C-A¹ V T¹-L¹-I-L²-T²-A¹ VI T¹-L¹-I-L²-T²-C-A¹ VIIT¹-L¹-T²-L²-T³-A¹ VIII T¹-L¹-T²-L²-T³-C-A¹ IX T¹-P¹-I-P²-T²-A¹ XT¹-P¹-I-P²-T²-C-A¹ XI T¹-P¹-T²-P²-T³-A¹ XII T¹-P¹-T²-P²-T³-C-A¹ XIIIT¹-P¹-L¹-I-L²-P²-T²-A¹ XIV T¹-P¹-L¹-I-L²-P²-T²-C-A¹ XVT¹-P¹-L¹-T²-L²-P²-T³-A¹ XVI T¹-P¹-L¹-T²-L²-P²-T³-C-A¹ XVIIT¹-L¹-P¹-T²-A¹ XVIII T¹-P¹-L¹-T²-A¹ XIX T¹-P¹-L¹-T²-C-A¹ XXT¹-P¹-I-P²-P³-T²-A¹ XXI T¹-P¹-I-P²-P³-T²-A¹ XXII T¹-P¹-I-T²-A¹ XXIIIT¹-P¹-I-T²-C-A¹ XXIV T¹-P¹-L-P²-P³-T²-A¹ XXV T¹-P¹-L-P²-P³-T²-C-A¹ XXVIT¹-P¹-T²-P²-P³-T³-A¹ XXVII T¹-P¹-T²-P²-P³-T³-C-A¹ XXVIII T¹-L-T²-A¹ XXIXT¹-L-T²-C-A¹ XXX T¹-I-T²-A¹ XXXI T¹-I-T²-C-A¹ XXXII T¹-P-T²-C-A¹

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof. Generally, the method comprisesadministering to the subject an amino-terminal immunoglobulin fusionprotein comprising a therapeutic peptide such as a relaxin peptideattached to the amino terminus of an immunoglobulin region. In someembodiments, an immunoglobulin fusion protein having the formula of I,II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII,XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII, XXIX,XXX, XXXI, XXXII, or any modification, portions, or additions thereof isadministered to a patient. In some embodiments, one or more of theimmunoglobulin fusion proteins I, II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII,XXIV, XXV, XXVI, XXVII, XXVIII, XXIX, XXX, XXXI, or XXXII, furthercomprising a second immunoglobulin region, is administered to a patient.

Further disclosed herein are methods of improving the delivery of atherapeutic peptide. The methods may involve generation of anamino-terminal immunoglobulin fusion protein from a genetic construct.In some embodiments, the immunoglobulin fusion protein is recombinantlyproduced from a genetic construct encoding the immunoglobulin fusionprotein. In some embodiments, the construct is expressed in vitro usingstandard mammalian cell culture techniques. In some embodiments, oneconstruct encoding a therapeutic peptide connected to the amino-terminusof a first immunoglobulin region is co-expressed with a second constructcomprising a second immunoglobulin region, to produce a recombinantimmunoglobulin fusion protein. In some embodiments, a construct encodinga protease is co-expressed with an immunoglobulin fusion protein. Themethod may further comprise generating immunoglobulin genetic fusionconstructs comprising one or more connecting peptides, internal linkers,linkers, and/or proteolytic cleavage sites.

Before the present methods and compositions are described, it is to beunderstood that this disclosure is not limited to a particular method orcomposition described, and as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting.Examples are put forth so as to provide those of ordinary skill in theart with a disclosure and description of how to make and use theimmunoglobulin fusion proteins provided herein, and are not intended tolimit the scope of what the inventors regard as their invention nor arethey intended to represent that the provided experiments encompass allof the experiments performed. Efforts have been made to ensure accuracywith respect to numbers used but some experimental errors and deviationsshould be accounted for.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited. It is understood that the present disclosure supersedes anydisclosure of an incorporated publication to the extent there is acontradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible. Any recitedcombination of amino acid sequences can have the order recited, or anyother order which is logically possible. As a non-limiting example, animmunoglobulin fusion protein comprising an insulin therapeutic peptide,T, and an immunoglobulin region, A, includes, for example and withoutlimitation: T-A, A-T, T-A-T, and A-T-A.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The terms “homologous,” “homology,” or “percent homology” when usedherein to describe to an amino acid sequence or a nucleic acid sequence,relative to a reference sequence, can be determined using the formuladescribed by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877,1993). Such a formula is incorporated into the basic local alignmentsearch tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent homology of sequences can be determined usingthe most recent version of BLAST, as of the filing date of thisapplication.

Relaxin Immunoglobulin Fusion Proteins

Various insulin immunoglobulin fusion proteins disclosed herein comprisea first immunoglobulin region and a relaxin therapeutic peptide, whereinthe relaxin therapeutic peptide, or an amino acid sequence thereof, isconnected to an amino terminus of the first immunoglobulin region. Invarious instances, the insulin immunoglobulin fusion proteins furthercomprise a second immunoglobulin region. The immunoglobulin region(first and/or second) may be any portion, in part or whole, of animmunoglobulin.

The immunoglobulin region may comprise an entire immunoglobulin moleculeor any polypeptide comprising a fragment of an immunoglobulin including,but not limited to, heavy chain, light chain, variable domain, constantdomain, complementarity determining region (CDR), framework region,fragment antigen binding (Fab) region, Fab′, F(ab′)2, F(ab′)3, Fab′,fragment crystallizable (Fc) region, single chain variable fragment(scFV), di-scFv, single domain immunoglobulin, trifunctionalimmunoglobulin, chemically linked F(ab′)2, and any portion orcombination thereof. In some embodiments, an immunoglobulin heavy chainmay comprise an entire heavy chain or a portion of a heavy chain. Forexample, a variable domain or region thereof derived from a heavy chainmay be referred to as a heavy chain or a region of a heavy chain. Insome embodiments, an immunoglobulin light chain may comprise an entirelight chain or a portion of a light chain. For example, a variabledomain or region thereof derived from a light chain may be referred toas a light chain or a region of a light chain. The immunoglobulin regionmay be bispecific or trispecific. A single domain immunoglobulinincludes, but is not limited to, a single monomeric variableimmunoglobulin domain. The single domain immunoglobulin may be a sharkvariable new antigen receptor immunoglobulin fragment (VNAR). Theimmunoglobulin may be derived from any type known to one of skill in theart including, but not limited to, IgA, IgD, IgE, IgG, IgM, IgY, IgW.The immunoglobulin region may be a glycoprotein. The immunoglobulinregion may comprise one or more functional units, including but notlimited to, 1, 2, 3, 4, and 5 units. The immunoglobulin region maycomprise one or more units connected by one or more disulfide bonds. Theimmunoglobulin region may comprise one or more units connected by apeptide linker, for example, a scFv immunoglobulin. The immunoglobulinmay be a recombinant immunoglobulin including immunoglobulins with aminoacid mutations, substitutions, and/or deletions. The immunoglobulin maybe a recombinant immunoglobulin comprising chemical modifications. Theimmunoglobulin may comprise a whole or part of an immunoglobulin-drugconjugate. The immunoglobulin may comprise a small molecule. Theimmunoglobulin may comprise a whole or part of an immunoglobulin-drugconjugate comprising a small molecule. The immunoglobulin may be from amammalian source. The immunoglobulin may be a chimeric immunoglobulin.The immunoglobulin region may be derived in whole or in part from anengineered immunoglobulin or recombinant immunoglobulin. Theimmunoglobulin may be from a humanized, human engineered or fully humanimmunoglobulin. The mammalian immunoglobulin may be a bovineimmunoglobulin. The mammalian immunoglobulin may be a humanimmunoglobulin. The mammalian immunoglobulin may be a murineimmunoglobulin. The mammalian immunoglobulin may be a non-human primateimmunoglobulin. The immunoglobulin may be an avian immunoglobulin. Theimmunoglobulin may be a shark immunoglobulin.

The immunoglobulin heavy chain fusion may comprise an amino acidsequence that is based on or derived from any one of SEQ ID NOs: 13-16,20-22. The immunoglobulin heavy chain fusion may comprise an amino acidsequence that is at least about 50% homologous to any one of SEQ ID NOs:13-16, 20-22. The immunoglobulin heavy chain fusion may comprise anamino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% homologous to any one of SEQ ID NOs: 13-16, 20-22. Theimmunoglobulin heavy chain fusion may comprise an amino acid sequencethat is at least about 70% homologous to any one of SEQ ID NOs: 13-16,20-22. The immunoglobulin heavy chain fusion may comprise an amino acidsequence that is at least about 80% homologous to any one of SEQ ID NOs:13-16, 20-22. The immunoglobulin heavy chain fusion may comprise anamino acid sequence that is at least about 50% identical to any one ofSEQ ID NOs: 13-16, 20-22. The immunoglobulin heavy chain fusion maycomprise an amino acid sequence that is at least about 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 97% identical to any one of SEQ ID NOs:13-16, 20-22. The immunoglobulin heavy chain fusion may comprise anamino acid sequence that is at least about 70% identical to any one ofSEQ ID NOs: 13-16, 20-22. The immunoglobulin heavy chain fusion maycomprise an amino acid sequence that is at least about 80% identical toany one of SEQ ID NOs: 13-16, 20-22. The immunoglobulin heavy chainfusion may comprise an amino acid sequence that is 100% identical to anyone of SEQ ID NOs: 13-16, 20-22. The immunoglobulin light chain maycomprise an amino acid sequence that is based on or derived from any oneof SEQ ID NOs: 9-12. The immunoglobulin light chain may comprise anamino acid sequence that is at least about 50% homologous to any one ofSEQ ID NOs: 9-12. The immunoglobulin light chain may comprise an aminoacid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 97% homologous to any one of SEQ ID NOs: 9-12. Theimmunoglobulin light chain may comprise an amino acid sequence that isat least about 70% homologous to any one of SEQ ID NOs: 9-12. Theimmunoglobulin light chain may comprise an amino acid sequence that isat least about 80% homologous to any one of SEQ ID NOs: 9-12. Theimmunoglobulin light chain may comprise an amino acid sequence that isat least about 50% identical to any one of SEQ ID NOs: 9-12. Theimmunoglobulin light chain may comprise an amino acid sequence that isat least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% identicalto any one of SEQ ID NOs: 9-12. The immunoglobulin light chain maycomprise an amino acid sequence that is at least about 70% identical toany one of SEQ ID NOs: 9-12. The immunoglobulin light chain may comprisean amino acid sequence that is at least about 80% identical to any oneof SEQ ID NOs: 9-12. The immunoglobulin light chain may comprise anamino acid sequence that is 100% identical to any one of SEQ ID NOs:9-12.

The immunoglobulin heavy chain fusion may comprise an amino acidsequence that is based on or derived from SEQ ID NO: 22. Theimmunoglobulin heavy chain fusion may comprise an amino acid sequencethat is at least about 50% homologous to SEQ ID NO: 22. Theimmunoglobulin heavy chain fusion may comprise an amino acid sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97%homologous to SEQ ID NO: 22. The immunoglobulin heavy chain fusion maycomprise an amino acid sequence that is at least about 70% homologous toSEQ ID NO: 22. The immunoglobulin heavy chain fusion may comprise anamino acid sequence that is at least about 80% homologous to SEQ ID NO:22. The immunoglobulin heavy chain fusion may comprise an amino acidsequence that is at least about 50% identical to SEQ ID NO: 22. Theimmunoglobulin heavy chain fusion may comprise an amino acid sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97%identical to SEQ ID NO: 22. The immunoglobulin heavy chain fusion maycomprise an amino acid sequence that is at least about 70% identical toSEQ ID NO: 22. The immunoglobulin heavy chain fusion may comprise anamino acid sequence that is at least about 80% identical to SEQ ID NO:22. The immunoglobulin heavy chain fusion may comprise an amino acidsequence that is 100% identical to SEQ ID NO: 22. The immunoglobulinlight chain may comprise an amino acid sequence that is based on orderived from SEQ ID NO: 12. The immunoglobulin light chain may comprisean amino acid sequence that is at least about 50% homologous to SEQ IDNO: 12. The immunoglobulin light chain may comprise an amino acidsequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or 97% homologous to SEQ ID NO: 12. The immunoglobulin light chain maycomprise an amino acid sequence that is at least about 70% homologous toSEQ ID NO: 12. The immunoglobulin light chain may comprise an amino acidsequence that is at least about 80% homologous to SEQ ID NO: 12. Theimmunoglobulin light chain may comprise an amino acid sequence that isat least about 50% identical to SEQ ID NO: 12. The immunoglobulin lightchain may comprise an amino acid sequence that is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% identical to SEQ ID NO: 12.The immunoglobulin light chain may comprise an amino acid sequence thatis at least about 70% identical to SEQ ID NO: 12. The immunoglobulinlight chain may comprise an amino acid sequence that is at least about80% identical to SEQ ID NO: 12. The immunoglobulin light chain maycomprise an amino acid sequence that is 100% identical to SEQ ID NO: 12.

The immunoglobulin heavy chain fusion may comprise an amino acidsequence comprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or moreamino acids based on or derived from any one of SEQ ID NOs: 13-16,20-22. The immunoglobulin heavy chain fusion may comprise an amino acidsequence comprising 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 450, 500 or more amino acids based on orderived from any one of SEQ ID NOs: 13-16, 20-22. The immunoglobulinheavy chain fusion may comprise an amino acid sequence comprising 10 ormore amino acids based on or derived from any one of SEQ ID NOs: 13-16,20-22. The immunoglobulin heavy chain fusion may comprise an amino acidsequence comprising 50 or more amino acids based on or derived from anyone of SEQ ID NOs: 13-16, 20-22. The immunoglobulin heavy chain fusionmay comprise an amino acid sequence comprising 100 or more amino acidsbased on or derived from any one of SEQ ID NOs: 13-16, 20-22. Theimmunoglobulin heavy chain fusion may comprise an amino acid sequencecomprising 200 or more amino acids based on or derived from any one ofSEQ ID NOs: 13-16, 20-22. The amino acids may be consecutive.Alternatively, or additionally, the amino acids are nonconsecutive. Insome embodiments, the immunoglobulin heavy chain fusion may compriseamino acids derived from any one of SEQ ID NOs: 13-16, 20-22 and aminoacids not derived from any one of SEQ ID NOs: 13-16, 20-22. In someembodiments, the immunoglobulin heavy chain fusion may comprise aminoacids derived from one or more of SEQ ID NOs: 13-16, 20-22 and aminoacids not derived from any one of SEQ ID NOs: 13-16, 20-22. In someembodiments, the immunoglobulin heavy chain fusion comprises amino acidsderived from 1, 2, 3, 4, or 5 of SEQ ID NOs: 13-16, 20-22.

The immunoglobulin heavy chain fusion may be encoded by a nucleotidesequence that is based on or derived from any one of SEQ ID NOs: 5-8,17-19. The immunoglobulin heavy chain fusion may be encoded by anucleotide sequence that is at least about 50% homologous to any one ofSEQ ID NOs: 5-8, 17-19. The immunoglobulin heavy chain fusion may beencoded by a nucleotide sequence that is at least about 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 97% homologous to any one of SEQ ID NOs:5-8, 17-19. The immunoglobulin heavy chain fusion may be encoded by anucleotide sequence that is at least about 70% homologous to any one ofSEQ ID NOs: 5-8, 17-19. The immunoglobulin heavy chain fusion may beencoded by a nucleotide sequence that is at least about 80% homologousto any one of SEQ ID NOs: 5-8, 17-19. The immunoglobulin heavy chainfusion may be encoded by a nucleotide sequence that is at least about50% identical to any one of SEQ ID NOs: 5-8, 17-19. The immunoglobulinheavy chain fusion may be encoded by a nucleotide sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% identical toany one of SEQ ID NOs: 5-8, 17-19. The immunoglobulin heavy chain fusionmay be encoded by a nucleotide sequence that is at least about 70%identical to any one of SEQ ID NOs: 5-8, 17-19. The immunoglobulin heavychain fusion may be encoded by a nucleotide sequence that is at leastabout 80% identical to any one of SEQ ID NOs: 5-8, 17-19. Theimmunoglobulin heavy chain fusion may be encoded by a nucleotidesequence that is 100% identical to any one of SEQ ID NOs: 5-8, 17-19.

The immunoglobulin heavy chain fusion may be encoded by a nucleotidesequence comprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides based on or derived from any one of SEQ ID NOs: 5-8, 17-19.The immunoglobulin heavy chain fusion may be encoded by a nucleotidesequence comprising 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 450, 500 or more nucleotides based on orderived from any one of SEQ ID NOs: 5-8, 17-19. The immunoglobulin heavychain fusion may be encoded by a nucleotide sequence comprising 600,650, 700, 750, 800, 850, 900, 950, 1000 or more nucleotides based on orderived from any one of SEQ ID NOs: 5-8, 17-19. The immunoglobulin heavychain fusion may be encoded by a nucleotide sequence comprising 1100,1200, 1300, 1400, 1500 or more nucleotides based on or derived from anyone of SEQ ID NOs: 5-8, 17-19. The immunoglobulin heavy chain fusion maybe encoded by a nucleotide sequence comprising 100 or more nucleotidesbased on or derived from any one of SEQ ID NOs: 5-8, 17-19. Theimmunoglobulin heavy chain fusion may be encoded by a nucleotidesequence comprising 500 or more nucleotides based on or derived from anyone of SEQ ID NOs: 5-8, 17-19. The immunoglobulin heavy chain fusion maybe encoded by a nucleotide sequence comprising 1000 or more nucleotidesbased on or derived from any one of SEQ ID NOs: 5-8, 17-19. Theimmunoglobulin heavy chain fusion may be encoded by a nucleotidesequence comprising 1300 or more nucleotides based on or derived fromany one of SEQ ID NOs: 5-8, 17-19. The nucleotides may be consecutive.Alternatively, or additionally, the nucleotides are nonconsecutive. Insome embodiments, the immunoglobulin heavy chain fusion is encoded by anucleotide sequence comprising nucleotides derived from any one of SEQID NOs: 5-8, 17-19 and nucleotides not derived from any one of SEQ IDNOs: 5-8, 17-19. In some embodiments, the immunoglobulin heavy chainfusion is encoded by a nucleotide sequence comprising nucleotidesderived from one or more of SEQ ID NOs: 5-8, 17-19 and nucleotides notderived from any one of SEQ ID NOs: 5-8, 17-19. In some embodiments, theimmunoglobulin heavy chain fusion is encoded by a nucleotide sequencederived from 1, 2, 3, 4, or 5 of SEQ ID NOs: 5-8, 17-19.

Immunoglobulin Fusion Proteins

In one feature of the invention, provided herein are immunoglobulinfusion proteins comprising (a) an immunoglobulin light chain fusion, and(b) a second immunoglobulin region derived from an immunoglobulin heavychain, wherein the immunoglobulin light chain fusion is connected to thesecond immunoglobulin region by one or more disulfide bonds or aconnecting peptide. The immunoglobulin light chain fusion comprises afirst therapeutic peptide connected to the amino-terminus of a firstimmunoglobulin region derived from an immunoglobulin light chain. Insome embodiments, the second immunoglobulin region is attached to anon-immunoglobulin region, creating a second immunoglobulin fusion. Thenon-immunoglobulin region may comprise a second therapeutic peptide. Thenon-immunoglobulin region may comprise a linker peptide. Thenon-immunoglobulin region may comprise a proteolytic cleavage site. Thesecond therapeutic peptide may comprise an internal linker. In someembodiments, the second therapeutic peptide is attached to the amino- orcarboxyl-terminus of the second immunoglobulin region. In someembodiments, the second therapeutic peptide is attached to one or moreinternal amino acids of the second immunoglobulin region. In someembodiments, the second therapeutic peptide is attached to amino acidsof a loop portion within the second immunoglobulin region. In someembodiments, the therapeutic peptide is attached to the secondimmunoglobulin region using one or more linker peptides. Theimmunoglobulin light chain fusion may further comprise one or moreadditional therapeutic peptides.

In one feature of the invention, provided herein are immunoglobulinfusion proteins comprising (a) an immunoglobulin heavy chain fusion, and(b) a second immunoglobulin region derived from an immunoglobulin lightchain, wherein the immunoglobulin heavy chain fusion is connected to thesecond immunoglobulin region by one or more disulfide bonds or aconnecting peptide. The immunoglobulin heavy chain fusion comprises afirst therapeutic peptide connected to the amino-terminus of a firstimmunoglobulin region derived from an immunoglobulin heavy chain. Insome embodiments, the second immunoglobulin region is attached to anon-immunoglobulin region, creating a second immunoglobulin fusion. Thenon-immunoglobulin region may comprise a second therapeutic peptide. Thenon-immunoglobulin region may comprise a linker peptide. Thenon-immunoglobulin region may comprise a proteolytic cleavage site. Thesecond therapeutic peptide may comprise an internal linker. In someembodiments, the second therapeutic peptide is attached to the amino- orcarboxyl-terminus of the second immunoglobulin region. In someembodiments, the second therapeutic peptide is attached to one or moreinternal amino acids of the second immunoglobulin region. In someembodiments, the second therapeutic peptide is attached to amino acidsof a loop portion within the second immunoglobulin region. In someembodiments, the therapeutic peptide is attached to the secondimmunoglobulin region using one or more linker peptides. Theimmunoglobulin heavy chain fusion may further comprise one or moreadditional therapeutic peptides.

In one feature of the invention, provided herein are immunoglobulinfusion proteins comprising (a) an immunoglobulin light chain fusion, and(b) an immunoglobulin heavy chain fusion. The immunoglobulin light chainfusion comprises a first therapeutic peptide connected to theamino-terminus of a first immunoglobulin region derived from animmunoglobulin light chain. The immunoglobulin heavy chain fusioncomprises a first therapeutic peptide connected to the amino-terminus ofa first immunoglobulin region derived from an immunoglobulin heavychain. In some embodiments, the immunoglobulin light chain fusionfurther comprises one or more additional therapeutic peptides. In someembodiments, the immunoglobulin heavy chain fusion comprises one or moreadditional therapeutic peptides.

In one feature of the invention, provided herein are immunoglobulinfusion proteins comprising (a) an immunoglobulin light chain fusion, and(b) a second immunoglobulin region, wherein the immunoglobulin lightchain fusion comprises a first therapeutic peptide connected to theamino-terminus of a first immunoglobulin region derived from animmunoglobulin light chain. The second immunoglobulin region may bederived from an immunoglobulin heavy chain. The second immunoglobulinregion may be derived from an immunoglobulin light chain. The secondimmunoglobulin region may be connected to one or more non-immunoglobulinregions, creating a second immunoglobulin fusion. The non-immunoglobulinregion may comprise a second therapeutic peptide. The non-immunoglobulinregion may comprise a linker peptide. The non-immunoglobulin region maycomprise a proteolytic cleavage site. The second therapeutic peptide maycomprise an internal linker. In some embodiments, the second therapeuticpeptide is attached to the amino- or carboxyl-terminus of the secondimmunoglobulin region. In some embodiments, the second therapeuticpeptide is attached to one or more internal amino acids of the secondimmunoglobulin region. In some embodiments, the second therapeuticpeptide is attached to amino acids of a loop portion within the secondimmunoglobulin region. In some embodiments, the therapeutic peptide isattached to the second immunoglobulin region using one or more linkerpeptides. The immunoglobulin light chain fusion may further comprise oneor more additional therapeutic peptides.

In one feature of the invention, provided herein are immunoglobulinfusion proteins comprising (a) an immunoglobulin heavy chain fusion, and(b) a second immunoglobulin region, wherein the immunoglobulin heavychain fusion comprises a first therapeutic peptide connected to theamino-terminus of a first immunoglobulin region derived from animmunoglobulin heavy chain. The second immunoglobulin region may bederived from an immunoglobulin heavy chain. The second immunoglobulinregion may be derived from an immunoglobulin light chain. The secondimmunoglobulin region may be connected to one or more non-immunoglobulinregions, creating a second immunoglobulin fusion. The non-immunoglobulinregion may comprise a second therapeutic peptide. The non-immunoglobulinregion may comprise a linker peptide. The non-immunoglobulin region maycomprise a proteolytic cleavage site. The second therapeutic peptide maycomprise an internal linker. In some embodiments, the second therapeuticpeptide is attached to the amino- or carboxyl-terminus of the secondimmunoglobulin region. In some embodiments, the second therapeuticpeptide is attached to one or more internal amino acids of the secondimmunoglobulin region. In some embodiments, the second therapeuticpeptide is attached to amino acids of a loop portion within the secondimmunoglobulin region. In some embodiments, the therapeutic peptide isattached to the second immunoglobulin region using one or more linkerpeptides. The immunoglobulin heavy chain fusion may further comprise oneor more additional therapeutic peptides.

In one feature of the invention, provided herein is an immunoglobulinfusion protein comprising a relaxin therapeutic peptide connected to theamino-terminus of a region of an immunoglobulin heavy chain, wherein theimmunoglobulin fusion is referred to herein as an immunoglobulin heavychain fusion. In some embodiments, the immunoglobulin fusion proteinfurther comprises one or more regions of an immunoglobulin light chain,wherein the immunoglobulin heavy chain fusion is connected to the one ormore regions of an immunoglobulin light chain by disulfide bonds or aconnecting peptide. In some embodiments, the therapeutic peptidecomprises a relaxin B chain and a relaxin A chain. The relaxin B andrelaxin A chains may be connected by a peptide linker. The peptidelinker may comprise a protease cleavage site.

The immunoglobulin fusion protein may comprise an amino acid sequencethat is based on or derived from any one of SEQ ID NOs: 20-22. Theimmunoglobulin fusion protein may comprise an amino acid sequence thatis at least about 50% homologous to any one of SEQ ID NOs: 20-22. Theimmunoglobulin fusion protein may comprise an amino acid sequence thatis at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97%homologous to any one of SEQ ID NOs 20-22. The immunoglobulin fusionprotein may comprise an amino acid sequence that is at least about 70%homologous to any one of SEQ ID NOs: 20-22. The immunoglobulin fusionprotein may comprise an amino acid sequence that is at least about 80%homologous to any one of SEQ ID NOs: 20-22. The immunoglobulin fusionprotein may comprise an amino acid sequence that is at least about 50%identical to any one of SEQ ID NOs: 20-22. The immunoglobulin fusionprotein may comprise an amino acid sequence that is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% identical to any one of SEQ IDNOs 20-22. The immunoglobulin fusion protein may comprise an amino acidsequence that is at least about 70% identical to any one of SEQ ID NOs:20-22. The immunoglobulin fusion protein may comprise an amino acidsequence that is at least about 80% identical to any one of SEQ ID NOs:20-22. The immunoglobulin fusion protein may comprise an amino acidsequence that is 100% identical to any one of SEQ ID NOs: 20-22. In someembodiments, the immunoglobulin fusion protein comprises an amino acidsequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% homologous to an amino acid sequence of any one of SEQID NOs: 20-22. In some embodiments, the immunoglobulin fusion proteincomprises an amino acid sequence that is at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% identical to an amino acidsequence of any one of SEQ ID NOs: 20-22.

The immunoglobulin fusion protein may comprise an amino acid sequencethat is based on or derived from SEQ ID NO: 22. The immunoglobulinfusion protein may comprise an amino acid sequence that is at leastabout 50% homologous to SEQ ID NO: 22. The immunoglobulin fusion proteinmay comprise an amino acid sequence that is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to SEQ ID NO: 22. Theimmunoglobulin fusion protein may comprise an amino acid sequence thatis at least about 70% homologous to SEQ ID NO: 22. The immunoglobulinfusion protein may comprise an amino acid sequence that is at leastabout 80% homologous to SEQ ID NO: 22. The immunoglobulin fusion proteinmay comprise an amino acid sequence that is at least about 50% identicalto SEQ ID NO: 22. The immunoglobulin fusion protein may comprise anamino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% identical to SEQ ID NO: 22. The immunoglobulin fusionprotein may comprise an amino acid sequence that is at least about 70%identical to SEQ ID NO: 22. The immunoglobulin fusion protein maycomprise an amino acid sequence that is at least about 80% identical toSEQ ID NO: 22. The immunoglobulin fusion protein may comprise an aminoacid sequence that is 100% identical to SEQ ID NO: 22. In someembodiments, the immunoglobulin fusion protein comprises an amino acidsequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% homologous to an amino acid sequence of SEQ ID NO: 22.In some embodiments, the immunoglobulin fusion protein comprises anamino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 97% identical to an amino acid sequence of SEQ IDNO: 22.

The immunoglobulin fusion protein may comprise an amino acid sequencecomprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acidsbased on or derived from any one of SEQ ID NOs: 20-22. Theimmunoglobulin fusion protein may comprise an amino acid sequencecomprising 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 450, 500 or more amino acids based on or derived from anyone of SEQ ID NOs: 20-22. The immunoglobulin fusion protein may comprisean amino acid sequence comprising 10 or more amino acids based on orderived from any one of SEQ ID NOs: 20-22. The immunoglobulin fusionprotein may comprise an amino acid sequence comprising 50 or more aminoacids based on or derived from any one of SEQ ID NOs: 20-22. Theimmunoglobulin fusion protein may comprise an amino acid sequencecomprising 100 or more amino acids based on or derived from any one ofSEQ ID NOs: 20-22. The immunoglobulin fusion protein may comprise anamino acid sequence comprising 200 or more amino acids based on orderived from any one of SEQ ID NOs: 20-22. The amino acids may beconsecutive. Alternatively, or additionally, the amino acids arenonconsecutive. In some embodiments, the immunoglobulin fusion proteinmay comprise amino acids derived from any one of SEQ ID NOs: 20-22 andamino acids not derived from any one of SEQ ID NOs: 20-22. In someembodiments, the immunoglobulin fusion protein may comprise amino acidsderived from one or more of SEQ ID NOs: 20-22 and amino acids notderived from any one of SEQ ID NOs: 20-22. In some embodiments, theimmunoglobulin fusion protein comprises amino acids derived from 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more of SEQ ID NOs: 20-22.

The immunoglobulin fusion protein may be encoded by a nucleotidesequence that is based on or derived from any one of SEQ ID NOs: 17-19.The immunoglobulin fusion protein may be encoded by a nucleotidesequence that is at least about 50% homologous to any one of SEQ ID NOs:17-19. The immunoglobulin fusion protein may be encoded by a nucleotidesequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or 97% homologous to any one of SEQ ID NOs: 17-19. The immunoglobulinfusion protein may be encoded by a nucleotide sequence that is at leastabout 70% homologous to any one of SEQ ID NOs: 17-19. The immunoglobulinfusion protein may be encoded by a nucleotide sequence that is at leastabout 80% homologous to any one of SEQ ID NOs: 17-19. The immunoglobulinfusion protein may be encoded by a nucleotide sequence that is at leastabout 50% identical to any one of SEQ ID NOs: 17-19. The immunoglobulinfusion protein may be encoded by a nucleotide sequence that is at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% identical to anyone of SEQ ID NOs: 17-19. The immunoglobulin fusion protein may beencoded by a nucleotide sequence that is at least about 70% identical toany one of SEQ ID NOs: 17-19. The immunoglobulin fusion protein may beencoded by a nucleotide sequence that is at least about 80% identical toany one of SEQ ID NOs: 17-19. The immunoglobulin fusion protein may beencoded by a nucleotide sequence that is 100% identical to any one ofSEQ ID NOs: 17-19. In some embodiments, the immunoglobulin fusionprotein is encoded by a nucleotide sequence that is at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to anamino acid sequence of any one of SEQ ID NOs: 17-19. In someembodiments, the immunoglobulin fusion protein is encoded by anucleotide sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 97% identical to an amino acid sequence of anyone of SEQ ID NOs: 17-19.

The immunoglobulin fusion protein may be encoded by a nucleotidesequence that is based on or derived from SEQ ID NO: 19. Theimmunoglobulin fusion protein may be encoded by a nucleotide sequencethat is at least about 50% homologous to SEQ ID NO: 19. Theimmunoglobulin fusion protein may be encoded by a nucleotide sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97%homologous to SEQ ID NO: 19. The immunoglobulin fusion protein may beencoded by a nucleotide sequence that is at least about 70% homologousto SEQ ID NO: 19. The immunoglobulin fusion protein may be encoded by anucleotide sequence that is at least about 80% homologous to SEQ ID NO:19. The immunoglobulin fusion protein may be encoded by a nucleotidesequence that is at least about 50% identical to SEQ ID NO: 19. Theimmunoglobulin fusion protein may be encoded by a nucleotide sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97%identical to SEQ ID NO: 19. The immunoglobulin fusion protein may beencoded by a nucleotide sequence that is at least about 70% identical toSEQ ID NO: 19. The immunoglobulin fusion protein may be encoded by anucleotide sequence that is at least about 80% identical to SEQ ID NO:19. The immunoglobulin fusion protein may be encoded by a nucleotidesequence that is 100% identical to SEQ ID NO: 19. In some embodiments,the immunoglobulin fusion protein is encoded by a nucleotide sequencethat is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or 97% homologous to SEQ ID NO: 19. In some embodiments, theimmunoglobulin fusion protein is encoded by a nucleotide sequence thatis at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or97% identical to SEQ ID NO: 19.

The immunoglobulin fusion protein may be encoded by a nucleotidesequence comprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides based on or derived from any one of SEQ ID NOs: 17-19. Theimmunoglobulin fusion protein may be encoded by a nucleotide sequencecomprising 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 450, 500 or more nucleotides based on or derived from anyone of SEQ ID NOs: 17-19. The immunoglobulin fusion protein may beencoded by a nucleotide sequence comprising 600, 650, 700, 750, 800,850, 900, 950, 1000 or more nucleotides based on or derived from any oneof SEQ ID NOs: 17-19. The immunoglobulin fusion protein may be encodedby a nucleotide sequence comprising 1100, 1200, 1300, 1400, 1500 or morenucleotides based on or derived from any one of SEQ ID NOs: 17-19. Theimmunoglobulin fusion protein may be encoded by a nucleotide sequencecomprising 100 or more nucleotides based on or derived from any one ofSEQ ID NOs: 17-19. The immunoglobulin fusion protein may be encoded by anucleotide sequence comprising 500 or more nucleotides based on orderived from any one of SEQ ID NOs: 17-19. The immunoglobulin fusionprotein may be encoded by a nucleotide sequence comprising 1,000 or morenucleotides based on or derived from any one of SEQ ID NOs: 17-19. Theimmunoglobulin fusion protein may be encoded by a nucleotide sequencecomprising 1,300 or more nucleotides based on or derived from any one ofSEQ ID NOs: 17-19. The nucleotides may be consecutive. Alternatively, oradditionally, the nucleotides are nonconsecutive. In some embodiments,the immunoglobulin fusion protein is encoded by a nucleotide sequencecomprising nucleotides derived from any one of SEQ ID NOs: 17-19 andnucleotides not derived from any one of SEQ ID NOs: 17-19. In someembodiments, the immunoglobulin fusion protein is encoded by anucleotide sequence comprising nucleotides derived from one or more ofSEQ ID NOs: 25-44 and nucleotides not derived from any one of SEQ IDNOs: 17-19. In some embodiments, the immunoglobulin fusion protein isencoded by a nucleotide sequence derived from 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more of SEQ ID NOs: 17-19.

Further disclosed herein are nucleotide constructs comprising anucleotide sequence that is based on or derived from any one of SEQ IDNOs: 17-19. The nucleotide construct may be a plasmid for expression ina host cell. For example, a mammalian or bacterial expression plasmid.In some embodiments, the construct comprises a nucleotide sequence thatis at least about 50% homologous to any one of SEQ ID NOs: 17-19. Insome embodiments, the construct comprises a nucleotide sequence that isat least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologousto any one of SEQ ID NOs: 17-19. In some embodiments, the constructcomprises a nucleotide sequence that is at least about 70% homologous toany one of SEQ ID NOs: 17-19. In some embodiments, the constructcomprises a nucleotide sequence that is at least about 80% homologous toany one of SEQ ID NOs: 17-19. In some embodiments, the constructcomprises a nucleotide sequence that is at least about 50% identical toany one of SEQ ID NOs: 17-19. In some embodiments, the constructcomprises a nucleotide sequence that is at least about 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 97% identical to any one of SEQ ID NOs:17-19. In some embodiments, the construct comprises a nucleotidesequence that is at least about 70% identical to any one of SEQ ID NOs:17-19. In some embodiments, the construct comprises a nucleotidesequence that is at least about 80% identical to any one of SEQ ID NOs:17-19. In some embodiments, the construct comprises a nucleotidesequence that is 100% identical to any one of SEQ ID NOs: 17-19. In someembodiments, the construct comprises a nucleotide sequence that is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97%homologous to an amino acid sequence of any one of SEQ ID NOs: 17-19. Insome embodiments, the construct comprises a nucleotide sequence that isat least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97%identical to an amino acid sequence of any one of SEQ ID NOs: 17-19.

Further disclosed herein are nucleotide constructs comprising anucleotide sequence that is based on or derived from SEQ ID NO: 19. Thenucleotide construct may be a plasmid for expression in a host cell. Forexample, a mammalian or bacterial expression plasmid. In someembodiments, the construct comprises a nucleotide sequence that is atleast about 50% homologous to SEQ ID NO: 19. In some embodiments, theconstruct comprises a nucleotide sequence that is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to SEQ ID NO: 19.In some embodiments, the construct comprises a nucleotide sequence thatis at least about 70% homologous to SEQ ID NO: 19. In some embodiments,the construct comprises a nucleotide sequence that is at least about 80%homologous to SEQ ID NO: 19. In some embodiments, the constructcomprises a nucleotide sequence that is at least about 50% identical toa SEQ ID NO: 19. In some embodiments, the construct comprises anucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% identical to SEQ ID NO: 19. In some embodiments, theconstruct comprises a nucleotide sequence that is at least about 70%identical to SEQ ID NO: 19. In some embodiments, the construct comprisesa nucleotide sequence that is at least about 80% identical to SEQ ID NO:19. In some embodiments, the construct comprises a nucleotide sequencethat is 100% identical to SEQ ID NO: 19. In some embodiments, theconstruct comprises a nucleotide sequence that is at least about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to SEQ IDNO: 19. In some embodiments, the construct comprises a nucleotidesequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% identical to SEQ ID NO: 19.

The immunoglobulin fusion protein may comprise an immunoglobulin heavychain fusion that is based on or derived from any one or more of SEQ IDNOs: 20-22.

The immunoglobulin fusion protein may comprise an immunoglobulin heavychain fusion that is based on or derived from SEQ ID NO: 22.

The immunoglobulin fusion protein may comprise a second immunoglobulinregion derived from an immunoglobulin heavy chain including any one ormore of SEQ ID NOs: 20-22.

The immunoglobulin fusion protein may comprise an immunoglobulin lightchain fusion that is based on or derived from any one or more of SEQ IDNOs: 9-12.

The immunoglobulin fusion protein may comprise an immunoglobulin lightchain fusion that is based on or derived from SEQ ID NO: 12.

The immunoglobulin fusion protein may comprise a second immunoglobulinregion derived from an immunoglobulin light chain including any one ormore of SEQ ID NOs: 9-12.

The immunoglobulin fusion protein may comprise (a) a region of animmunoglobulin heavy chain that is based on or derived from any one ormore of SEQ ID NOs: 20-22; and (b) a region of an immunoglobulin lightchain that is based on or derived from any one or more of SEQ ID NOs:9-12. The immunoglobulin fusion protein may comprise (a) a region of animmunoglobulin heavy chain comprising an amino acid sequence that is atleast about 50% identical to SEQ ID NOs 20-22; and (b) a region of animmunoglobulin light chain comprising an amino acid sequence that is atleast about 50% identical to SEQ ID NOs: 9-12. The region of animmunoglobulin heavy chain may comprise an amino acid sequence that isat least about 60%, 70%, 75%, 80%, 90%, 95%, or 97% identical to SEQ IDNOs: 20-22. The region of an immunoglobulin heavy chain may comprise anamino acid sequence that is 100% identical to SEQ ID NOs: 20-22. Theregion of an immunoglobulin light chain may comprise an amino acidsequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, or 97%identical to SEQ ID NOs: 9-12. The region of an immunoglobulin lightchain may comprise an amino acid sequence that is 100% identical to SEQID NOs: 9-12.

The immunoglobulin fusion protein may comprise (a) a region of animmunoglobulin heavy chain that is based on or derived from SEQ ID NO:22; and (b) a region of an immunoglobulin light chain that is based onor derived from SEQ ID NO: 12. The immunoglobulin fusion protein maycomprise (a) a region of an immunoglobulin heavy chain comprising anamino acid sequence that is at least about 50% identical to SEQ ID NO:22; and (b) a region of an immunoglobulin light chain comprising anamino acid sequence that is at least about 50% identical to SEQ ID NO:12. The region of an immunoglobulin heavy chain may comprise an aminoacid sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%, or97% identical to SEQ ID NO: 22. The region of an immunoglobulin heavychain may comprise an amino acid sequence that is 100% identical to SEQID NO: 22. The region of an immunoglobulin light chain may comprise anamino acid sequence that is at least about 60%, 70%, 75%, 80%, 90%, 95%,or 97% identical to SEQ ID NO: 12. The region of an immunoglobulin lightchain may comprise an amino acid sequence that is 100% identical to SEQID NO: 12.

The immunoglobulin fusion protein may comprise (a) a region of animmunoglobulin heavy chain encoded by a nucleotide sequence of SEQ IDNOs: 5-8, 17-19; and (b) a region of an immunoglobulin light chainencoded by a nucleotide sequence of SEQ ID NOs: 1-4. The immunoglobulinprotein may comprise (a) a region of an immunoglobulin heavy chainencoded by a nucleotide sequence that is at least 50% or more identicalto a nucleotide sequence of SEQ ID NOs: 5-8, 17-19; and (b) a region ofan immunoglobulin light chain encoded by a nucleotide sequence that isat least 50% or more identical to a nucleotide sequence of SEQ ID NOs:1-4. The region of an immunoglobulin heavy chain may be encoded by anucleotide sequence that is at least 60%, 70%, 75%, 80%, 90%, 95%, or97% or more identical to a nucleotide sequence of SEQ ID NOs: 5-8,17-19. The region of an immunoglobulin heavy chain may be encoded by anucleotide sequence that is 100% identical to a nucleotide sequence ofSEQ ID NOs: 5-8, 17-19. The region of an immunoglobulin light chain maybe encoded by a nucleotide sequence that is at least 60%, 70%, 75%, 80%,90%, 95%, or 97% or more identical to a nucleotide sequence of SEQ IDNOs: 1-4. The region of an immunoglobulin light chain may be encoded bya nucleotide sequence that is 100% identical to a nucleotide sequence ofSEQ ID NOs: 1-4.

The immunoglobulin glucagon fusion protein may comprise (a) a firstimmunoglobulin fusion protein encoded by a nucleotide sequence of anyone of SEQ ID NOs: 17-19; and (b) a second immunoglobulin proteinencoded by a nucleotide sequence of SEQ ID NO: 1. The immunoglobulinglucagon fusion protein may comprise (a) a first immunoglobulin fusionprotein encoded by a nucleotide sequence that is at least 50% or morehomologous to a nucleotide sequence of any one of SEQ ID NOs: 17-19; and(b) a second immunoglobulin protein encoded by a nucleotide sequencethat is at least 50% or more homologous to a nucleotide sequence of SEQID NO: 1. In some embodiments, the first immunoglobulin fusion proteinis encoded by a nucleotide sequence that is at least 60%, 70%, 75%, 80%,90%, 95%, or 97% or more homologous to a nucleotide sequence of any oneof SEQ ID NOs: 17-19. In some embodiments, the second immunoglobulinprotein is encoded by a nucleotide sequence that is at least 60%, 70%,75%, 80%, 90%, 95%, or 97% or more homologous to a nucleotide sequenceof SEQ ID NO: 1.

In some embodiments, provided herein are immunoglobulin relaxin fusionproteins. In some embodiments, the immunoglobulin relaxin fusionproteins comprise an immunoglobulin light chain and/or heavy chainregion fused at the amino terminus with a relaxin or a peptide derivedfrom relaxin, which includes relaxins having internal linkers. In someembodiments, the immunoglobulin relaxin fusion proteins further comprisea second immunoglobulin light chain and/or heavy chain. In someembodiments, an immunoglobulin relaxin fusion protein refers to a firstimmunoglobulin chain comprising an amino-terminal relaxin peptide orderivative thereof and a second immunoglobulin chain. In someembodiments, the first immunoglobulin relaxin fusion protein isco-expressed with the second immunoglobulin chain. In some embodiments,the immunoglobulin relaxin fusion protein comprises a heavy chain havingan amino acid sequence based on or derived from any one or more of SEQID NO: 20-22 and a light chain having an amino acid sequence based on orderived from any one or more of SEQ ID NO: 9-12. In some embodiments,the immunoglobulin relaxin fusion protein comprises a heavy chain havingan amino acid sequence based on or derived from SEQ ID NO: 22 and alight chain having an amino acid sequence based on or derived from SEQID NO: 12. In some embodiments, the immunoglobulin relaxin fusionproteins are configured to treat a disease or condition of the heart. Insome embodiments, the immunoglobulin relaxin fusion proteins treat adisease or condition including heart failure, acute coronary syndrome,atrial fibrillation, cardiac fibrosis, coronary artery disease, ischemiareperfusion associated with solid organ transplant (e.g., lung, kidney,liver, heart), cardiopulmonary bypass for organ protection (e.g.,renal), ischemic stroke, corneal healing (ocular administration),diabetic nephropathy, cirrhosis, portal hypertension, diabetic wouldhealing, systemic sclerosis, cervical ripening at time of labor,preeclampsia, portal hypertension, fibrosis, pancreatitis, inflammation,cancer, scleroderma, pulmonary fibrosis, renal fibrosis, hepaticfibrosis, thromboembolic disorders, reperfusion damage followingischemia, micro- and macrovascular lesions (vasculitis), arterial andvenous thromboses, edemas, ischemias such as myocardial infarction,stroke and transient ischemic attacks, for cardio protection inconnection with coronary artery bypass operations (coronary arterybypass graft, CABG), primary percutaneous transluminal coronaryangioplasties (PTCAs), PTCAs after thrombolysis, rescue PTCA, hearttransplants and open-heart operations, and for organ protection inconnection with transplants, bypass operations, catheter examinationsand other surgical procedures. Further provided is the use of thecompounds of the present disclosure for the prophylaxis and/or treatmentof respiratory disorders, such as, for example, chronic obstructivepulmonary disease (chronic bronchitis, COPD), interstitial lung disease,asthma, pulmonary emphysema, bronchiectases, cystic fibrosis(mucoviscidosis) and pulmonary hypertension, in particular pulmonaryarterial hypertension, and combinations thereof. In some embodiments,the immunoglobulin relaxin fusion proteins are configured to treat adisease or condition of the kidney. In some embodiments, theimmunoglobulin relaxin fusion proteins treat a disease or conditionincluding acute and chronic kidney diseases and acute and chronic renalinsufficiencies, as well as acute and chronic renal failure, includingacute and chronic stages of renal failure with and without therequirement of dialysis, as well as the underlying or related kidneydiseases such as renal hypoperfusion, dialysis induced hypotension,glomerulopathies, glomerular and tubular proteinuria, renal edema,hematuria, primary, secondary, as well as acute and chronicglomerulonephritis, membranous and membranoproliferativeglomerulonephritis, Alport-Syndrome, glomerulosclerosis, interstistialtubular diseases, nephropathic diseases, such as primary and inbornkidney diseases, renal inflammation, immunological renal diseases likerenal transplant rejection, immune complex induced renal diseases, aswell as intoxication induced nephropathic diseases, diabetic andnon-diabetic renal diseases, pyelonephritis, cystic kidneys,nephrosclerosis, hypertensive nephrosclerosis, nephrotic syndrome, thatare characterized and diagnostically associated with an abnormalreduction in creatinine clearance and/or water excretion, abnormalincreased blood concentrations of urea, nitrogen, potassium and/orcreatinine, alteration in the activity of renal enzymes, such asglutamylsynthetase, urine osmolarity and urine volume, increasedmicroalbuminuria, macroalbuminuria, glomerular and arteriolar lesions,tubular dilation, hyperphosphatemia, the requirement of dialysis, renalcarcinomas, after incomplete resection of the kidney, dehydration afteroveruse of diuretics, uncontrolled blood pressure increase withmalignant hypertension, urinary tract obstruction and infection,amyloidosis, as well as systemic diseases associated with glomerulardamage, such as Lupus erythematodes, and rheumatic immunologicalsystemic diseases, as well as renal artery stenosis, renal arterythrombosis, renal vein thrombosis, analgetics induced nephropathy, renaltubular acidosis, and combinations thereof. In some embodiments, theimmunoglobulin relaxin fusion proteins are configured to treat a diseaseor condition of the lung. In some embodiments the immunoglobulin relaxinfusion proteins treat a disease or condition including asthmaticdisorders, pulmonary arterial hypertension (PAH) and other forms ofpulmonary hypertension (PH) including left-heart disease, HIV, sicklecell anemia, thromboembolisms (CTEPH), sarcoidosis, COPD-associatedpulmonary hypertension, pulmonary fibrosis-associated pulmonaryhypertension, chronic-obstructive pulmonary disease (COPD), acuterespiratory distress syndrome (ARDS), acute lung injury (ALI),alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, pulmonaryemphysema (for example pulmonary emphysema induced by cigarette smoke),cystic fibrosis (CF), and combinations thereof.

Immunoglobulin Region

The immunoglobulin fusion proteins disclosed herein comprise one or moreimmunoglobulin regions. The immunoglobulin regions may comprise one ormore portions of an antigen binding domain, wherein the antigen bindingdomain is not specific for a human antigen. In some cases, theimmunoglobulin region is modified from a palivizumab antibody. Themodification may include one or more amino acid mutations, insertions ordeletions in one or more CDRs of the palivizumab antibody. As anon-limiting example, 1, 2, 3, 4 or 5 amino acids of the CDR3 of apalivizumab heavy chain sequence is modified.

The immunoglobulin region may comprise an entire immunoglobulin moleculeor any polypeptide comprising fragment of an immunoglobulin including,but not limited to, heavy chain, light chain, variable domain, constantdomain, complementarity determining region (CDR), framework region,fragment antigen binding (Fab) region, Fab′, F(ab′)2, F(ab′)3, Fab′,fragment crystallizable (Fc) region, single chain variable fragment(scFV), di-scFv, single domain immunoglobulin, trifunctionalimmunoglobulin, chemically linked F(ab′)2, and any combination thereof.In some embodiments, an immunoglobulin heavy chain may comprise anentire heavy chain or a portion of a heavy chain. For example, avariable domain or region thereof derived from a heavy chain may bereferred to as a heavy chain or a region of a heavy chain. In someembodiments, an immunoglobulin light chain may comprise an entire lightchain or a portion of a light chain. For example, a variable domain orregion thereof derived from a light chain may be referred to as a lightchain or a region of a light chain. A single domain immunoglobulinincludes, but is not limited to, a single monomeric variableimmunoglobulin domain, for example, a shark variable new antigenreceptor immunoglobulin fragment (VNAR).

The immunoglobulin may be derived from any type known to one of skill inthe art including, but not limited to, IgA, IgD, IgE, IgG, IgM, IgY,IgW. The immunoglobulin region may comprise one or more units, includingbut not limited to, 1, 2, 3, 4, and 5 units. Functional units mayinclude, but are not limited to, non-immunoglobulin regions, heavychain, light chain, variable domain, constant domain, complementaritydetermining region (CDR), framework region, fragment antigen binding(Fab) region, Fab′, F(ab′)2, F(ab′)3, Fab′, fragment crystallizable (Fc)region, single chain variable fragment (scFV), di-scFv, single domainimmunoglobulin, trifunctional immunoglobulin, chemically linked F(ab′)2,and any combination or fragments thereof. Non-immunoglobulin regionsinclude, but are not limited to, carbohydrates, lipids, small moleculesand therapeutic peptides. The immunoglobulin region may comprise one ormore units connected by one or more disulfide bonds. The immunoglobulinregion may comprise one or more units connected by a peptide linker, forexample, a scFv immunoglobulin. The immunoglobulin may be a recombinantimmunoglobulin including immunoglobulins with amino acid mutations,substitutions, and/or deletions. The immunoglobulin may be a recombinantimmunoglobulin comprising chemical modifications. The immunoglobulin maycomprise a whole or part of an immunoglobulin-drug conjugate.

The immunoglobulin region may comprise at least a portion of a humanimmunoglobulin. The immunoglobulin region may comprise at least aportion of a humanized immunoglobulin. The immunoglobulin region maycomprise at least a portion of a chimeric immunoglobulin. Theimmunoglobulin region may be based on or derived from a humanimmunoglobulin. The immunoglobulin region may be based on or derivedfrom a humanized immunoglobulin. The immunoglobulin region may be basedon or derived from a chimeric immunoglobulin. The immunoglobulin regionmay be based on or derived from a monoclonal immunoglobulin. Theimmunoglobulin region may be based on or derived from a polyclonalimmunoglobulin. The immunoglobulin region may comprise at least aportion of an immunoglobulin from a mammal, avian, reptile, amphibian,or a combination thereof. The mammal may be a human. The mammal may be anon-human primate. The mammal may be a dog, cat, sheep, goat, cow,rabbit, or mouse.

The immunoglobulin region may comprise at least a portion of animmunoglobulin heavy chain. The immunoglobulin region may comprise oneor more immunoglobulin heavy chains or a portion thereof. Theimmunoglobulin region may comprise two or more immunoglobulin heavychains or a portion thereof. The immunoglobulin region may comprise anamino acid sequence that is at least about 50% homologous to animmunoglobulin heavy chain. The immunoglobulin region may comprise anamino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, or 97% or more homologous to an immunoglobulin heavychain. The immunoglobulin region may comprise an amino acid sequencethat is at least about 70% homologous to an immunoglobulin heavy chain.The immunoglobulin region may comprise an amino acid sequence that is atleast about 80% homologous to an immunoglobulin heavy chain. Theimmunoglobulin region may comprise an amino acid sequence that is atleast about 90% homologous to an immunoglobulin heavy chain. Theimmunoglobulin heavy chain may comprise SEQ ID NOs: 13-16. In someembodiments, the immunoglobulin region comprises an amino acid sequencethat is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or 97% homologous to an amino acid sequence of any one of SEQ ID NOs:13-16. In some embodiments, the immunoglobulin region comprises an aminoacid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 97% identical to an amino acid sequence of any one ofSEQ ID NOs: 13-16.

The immunoglobulin region may comprise an amino acid sequence comprising5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or more aminoacids of an immunoglobulin heavy chain. The immunoglobulin region maycomprise an amino acid sequence comprising 100, 150, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900 or more amino acids of animmunoglobulin heavy chain. The amino acids may be consecutive.Alternatively, or additionally, the amino acids are non-consecutive.

The immunoglobulin heavy chain may be encoded by a nucleotide sequencebased on or derived from SEQ ID NOs: 5-8. The immunoglobulin heavy chainmay be encoded by a nucleotide sequence that is at least about 50%homologous to SEQ ID NOs: 5-8. The immunoglobulin heavy chain may beencoded by a nucleotide sequence that is at least about 60%, 65%, 70%,75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to SEQ ID NOs:5-8. The immunoglobulin heavy chain may be encoded by a nucleotidesequence that is at least about 75% homologous to SEQ ID NOs: 5-8. Theimmunoglobulin heavy chain may be encoded by a nucleotide sequence thatis at least about 85% homologous to SEQ ID NOs: 5-8. In someembodiments, the immunoglobulin region is encoded by a nucleotidesequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% homologous to a nucleotide sequence of any one of SEQID NOs: 5-8. In some embodiments, the immunoglobulin region is encodedby a nucleotide sequence that is at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 97% identical to a nucleotide sequence ofany one of SEQ ID NOs: 5-8.

The immunoglobulin region may comprise at least a portion of animmunoglobulin light chain. The immunoglobulin region may comprise oneor more immunoglobulin light chains or a portion thereof. Theimmunoglobulin region may comprise two or more immunoglobulin lightchains or a portion thereof. The immunoglobulin region may comprise anamino acid sequence that is at least about 50% homologous to animmunoglobulin light chain. The immunoglobulin region may comprise anamino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, or 97% or more homologous to an immunoglobulin lightchain. The immunoglobulin region may comprise an amino acid sequencethat is at least about 70% homologous to an immunoglobulin light chain.The immunoglobulin region may comprise an amino acid sequence that is atleast about 80% homologous to an immunoglobulin light chain. Theimmunoglobulin region may comprise an amino acid sequence that is atleast about 90% homologous to an immunoglobulin light chain. Theimmunoglobulin light chain may comprise SEQ ID NOs: 9-12. In someembodiments, the immunoglobulin region comprises an amino acid sequencethat is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or 97% homologous to an amino acid sequence of any one of SEQ ID NOs:9-12. In some embodiments, the immunoglobulin region comprises an aminoacid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 97% identical to an amino acid sequence of any one ofSEQ ID NOs: 9-12.

The immunoglobulin region may comprise an amino acid sequence comprising5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or more aminoacids of an immunoglobulin light chain. The immunoglobulin region maycomprise an amino acid sequence comprising 100, 150, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900 or more amino acids of animmunoglobulin light chain. The amino acids may be consecutive.Alternatively, or additionally, the amino acids are non-consecutive.

The immunoglobulin light chain may be encoded by a nucleotide sequencebased on or derived from SEQ ID NOs: 1-4. The immunoglobulin light chainmay be encoded by a nucleotide sequence that is at least about 50%homologous to SEQ ID NOs: 1-4. The immunoglobulin light chain may beencoded by a nucleotide sequence that is at least about 60%, 65%, 70%,75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to SEQ ID NOs:1-4. The immunoglobulin light chain may be encoded by a nucleotidesequence that is at least about 75% homologous to SEQ ID NOs: 1-4. Theimmunoglobulin light chain may be encoded by a nucleotide sequence thatis at least about 85% homologous to SEQ ID NOs: 1-4. In someembodiments, the immunoglobulin region is encoded by a nucleotidesequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% homologous to a nucleotide sequence of any one of SEQID NOs: 1-4. In some embodiments, the immunoglobulin region is encodedby a nucleotide sequence that is at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 97% identical to a nucleotide sequence ofany one of SEQ ID NOs: 1-4.

The immunoglobulin region may comprise at least a portion of a variabledomain. The immunoglobulin region may comprise one or more variabledomains or portions thereof. The immunoglobulin region may comprise 2,3, 4, 5 or more variable domains or portions thereof. The immunoglobulinregion may comprise an amino acid sequence comprising 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300,350, 400, 500 or more amino acids based on or derived from an amino acidsequence of one or more variable domains. The amino acids may beconsecutive. The amino acids may be non-consecutive.

The immunoglobulin region may comprise at least a portion of a constantdomain. The immunoglobulin region may comprise one or more constantdomains or portions thereof. The immunoglobulin region may comprise 2,3, 4, 5, 6, 7, 8, 9, 10 or more constant domains or portions thereof.The immunoglobulin region may comprise an amino acid sequence comprising10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225,250, 275, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400 ormore amino acids based on or derived from an amino acid sequence of oneor more constant domains. The amino acids may be consecutive. The aminoacids may be non-consecutive.

The immunoglobulin region may comprise at least a portion of acomplementarity-determining region (CDR). The immunoglobulin region maycomprise one or more complementarity-determining regions (CDRs) orportions thereof. The immunoglobulin region may comprise 2, 3, 4, 5 ormore complementarity-determining regions (CDRs) or portions thereof. Theimmunoglobulin region may comprise 6, 7, 8 or morecomplementarity-determining regions (CDRs) or portions thereof. Theimmunoglobulin region may comprise four or morecomplementarity-determining regions (CDRs) or portions thereof. Theimmunoglobulin region may comprise 9, 10, 11 or morecomplementarity-determining regions (CDRs) or portions thereof. The oneor more CDRs may be CDR1, CDR2, CDR3 or a combination thereof. The oneor more CDRs may be CDR1. The one or more CDRs may be CDR2. The one ormore CDRs may be CDR3. The CDR may be a heavy chain CDR. The one or moreCDRs may be a light chain CDR.

The immunoglobulin region may comprise a heavy chain CDR1 having anamino acid sequence based on or derived from SEQ ID NO: 72. Theimmunoglobulin region may comprise a heavy chain CDR2 having an aminoacid sequence based on or derived from SEQ ID NO: 73. The immunoglobulinregion may comprise a heavy chain CDR3 having an amino acid sequencebased on or derived from any one of SEQ ID NOs: 74-88. Theimmunoglobulin region may comprise a light chain CDR1 having an aminoacid sequence based on or derived from SEQ ID NO: 89. The immunoglobulinregion may comprise a light chain CDR2 having an amino acid sequencebased on or derived from SEQ ID NO: 90. The immunoglobulin region maycomprise a light chain CDR3 having an amino acid sequence based on orderived from any one of SEQ ID NOs: 91-97.

The immunoglobulin region may comprise a heavy chain CDR1 having anamino acid sequence that is at least about 50% homologous to SEQ ID NO:72. The immunoglobulin region may comprise a heavy chain CDR1 having anamino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% homologous to SEQ ID NO: 72. The immunoglobulin regionmay comprise a heavy chain CDR1 having an amino acid sequence that is atleast about 70% homologous to SEQ ID NO: 72. The immunoglobulin regionmay comprise a heavy chain CDR1 having an amino acid sequence that is atleast about 90% homologous to SEQ ID NO: 72. The immunoglobulin regionmay comprise a heavy chain CDR2 having an amino acid sequence that is atleast about 50% homologous to SEQ ID NO: 73. The immunoglobulin regionmay comprise a heavy chain CDR2 having an amino acid sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous toSEQ ID NO: 73. The immunoglobulin region may comprise a heavy chain CDR2having an amino acid sequence that is at least about 70% homologous toSEQ ID NO: 73. The immunoglobulin region may comprise a heavy chain CDR2having an amino acid sequence that is at least about 90% homologous toSEQ ID NO: 73. The immunoglobulin region may comprise a heavy chain CDR3having an amino acid sequence that is at least about 50% homologous toany one of SEQ ID NOs: 74-88. The immunoglobulin region may comprise aheavy chain CDR3 having an amino acid sequence that is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to any one ofSEQ ID NOs: 74-88. The immunoglobulin region may comprise a heavy chainCDR3 having an amino acid sequence that is at least about 70% homologousto any one of SEQ ID NOs: 74-88. The immunoglobulin region may comprisea heavy chain CDR3 having an amino acid sequence that is at least about90% homologous to any one of SEQ ID NOs: 74-88. The immunoglobulinregion may comprise a light chain CDR1 having an amino acid sequencethat is at least about 50% homologous to SEQ ID NO: 89. Theimmunoglobulin region may comprise a light chain CDR1 having an aminoacid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 97% homologous to SEQ ID NO: 89. The immunoglobulin region maycomprise a light chain CDR1 having an amino acid sequence that is atleast about 70% homologous to SEQ ID NO: 89. The immunoglobulin regionmay comprise a light chain CDR1 having an amino acid sequence that is atleast about 90% homologous to SEQ ID NO: 89. The immunoglobulin regionmay comprise a light chain CDR2 having an amino acid sequence that is atleast about 50% homologous to SEQ ID NO: 90. The immunoglobulin regionmay comprise a light chain CDR2 having an amino acid sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous toSEQ ID NO: 90. The immunoglobulin region may comprise a light chain CDR2having an amino acid sequence that is at least about 70% homologous toSEQ ID NO: 90. The immunoglobulin region may comprise a light chain CDR2having an amino acid sequence that is at least about 90% homologous toSEQ ID NO: 90. The immunoglobulin region may comprise a light chain CDR3having an amino acid sequence that is at least about 50% homologous toany one of SEQ ID NOs: 91-97. The immunoglobulin region may comprise alight chain CDR3 having an amino acid sequence that is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to any one ofSEQ ID NOs: 91-97. The immunoglobulin region may comprise a light chainCDR3 having an amino acid sequence that is at least about 70% homologousto any one of SEQ ID NOs: 91-97. The immunoglobulin region may comprisea light chain CDR3 having an amino acid sequence that is at least about90% homologous to any one of SEQ ID NOs: 91-97.

The immunoglobulin region may comprise an amino acid sequence comprising1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids based on or derivedfrom an amino acid sequence of a CDR. The immunoglobulin region maycomprise an amino acid sequence comprising 3 or more amino acids basedon or derived from an amino acid sequence of a CDR. The immunoglobulinregion may comprise an amino acid sequence comprising 5 or more aminoacids based on or derived from an amino acid sequence of a CDR. Theimmunoglobulin region may comprise an amino acid sequence comprising 10or more amino acids based on or derived from an amino acid sequence of aCDR. The amino acids may be consecutive. The amino acids may benon-consecutive.

The immunoglobulin region may be based on or derived from a palivizumabimmunoglobulin. The immunoglobulin region may comprise at least aportion of a palivizumab immunoglobulin. The immunoglobulin region maycomprise an amino acid sequence that is at least about 50% homologous toat least a portion of a palivizumab immunoglobulin. The immunoglobulinregion may comprise an amino acid sequence that is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more homologous to atleast a portion of a palivizumab immunoglobulin. The immunoglobulinregion may comprise an amino acid sequence that is at least about 70%homologous to at least a portion of a palivizumab immunoglobulin. Theimmunoglobulin region may comprise an amino acid sequence that is atleast about 80% homologous to at least a portion of a palivizumabimmunoglobulin.

The immunoglobulin region may comprise an amino acid sequence thatcomprises 10, 20, 30, 40, 50, 60, 70, 80, 90 or more amino acids of apalivizumab immunoglobulin sequence. The immunoglobulin region maycomprise an amino acid sequence that comprises 100, 200, 300, 400, 500,600, 700, 800, 900 or more amino acids of a palivizumab immunoglobulinsequence. The immunoglobulin region may comprise an amino acid sequencethat comprises 50 or more amino acids of a palivizumab immunoglobulinsequence. The immunoglobulin region may comprise an amino acid sequencethat comprises 100 or more amino acids of a palivizumab immunoglobulinsequence. The immunoglobulin region may comprise an amino acid sequencethat comprises 200 or more amino acids of a palivizumab immunoglobulinsequence.

The immunoglobulin region may comprise an amino acid sequence thatcomprises 10, 20, 30, 40, 50, 60, 70, 80, 90 or more consecutive aminoacids of a palivizumab immunoglobulin sequence. The immunoglobulinregion may comprise an amino acid sequence that comprises 100, 200, 300,400, 500, 600, 700, 800, 900 or more consecutive amino acids of apalivizumab immunoglobulin sequence. The immunoglobulin region maycomprise an amino acid sequence that comprises 50 or more consecutiveamino acids of a palivizumab immunoglobulin sequence. The immunoglobulinregion may comprise an amino acid sequence that comprises 100 or moreconsecutive amino acids of a palivizumab immunoglobulin sequence. Theimmunoglobulin region may comprise an amino acid sequence that comprises200 or more consecutive amino acids of a palivizumab immunoglobulinsequence.

The immunoglobulin region may comprise a CDR sequence based on orderived from a palivizumab CDR sequence. The immunoglobulin region maycomprise a palivizumab heavy chain CDR1 having an amino acid sequencebased on or derived from SEQ ID NO: 72. The immunoglobulin region maycomprise a palivizumab heavy chain CDR2 having an amino acid sequencebased on or derived from SEQ ID NO: 73. The immunoglobulin region maycomprise a palivizumab heavy chain CDR3 having an amino acid sequencebased on or derived from SEQ ID NO: 74. The immunoglobulin region maycomprise a mutant palivizumab heavy chain CDR3 having an amino acidsequence based on or derived from any one of SEQ ID NOs: 75-88. Theimmunoglobulin region having a mutant palivizumab heavy chain CDR3 mayhave reduced binding to RSV-F than wildtype palivizumab. Theimmunoglobulin region may comprise a palivizumab light chain CDR1 havingan amino acid sequence based on or derived from SEQ ID NO: 89. Theimmunoglobulin region may comprise a palivizumab light chain CDR2 havingan amino acid sequence based on or derived from SEQ ID NO: 90. Theimmunoglobulin region may comprise a palivizumab light chain CDR3 havingan amino acid sequence based on or derived from SEQ ID NO: 91. Theimmunoglobulin region may comprise a mutant palivizumab light chain CDR3having an amino acid sequence based on or derived from any one of SEQ IDNOs: 92-97. The immunoglobulin region having a mutant palivizumab lightchain CDR3 may have reduced binding to RSV-F than wildtype palivizumab.

The immunoglobulin region may comprise an amino acid sequence that isbased on or derived from any one of SEQ ID NOs: 10-14. Theimmunoglobulin region may comprise an amino acid sequence that is atleast about 50% homologous to any one of SEQ ID NOs: 10-14. Theimmunoglobulin region may comprise an amino acid sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous toany one of SEQ ID NOs 10-14. The immunoglobulin region may comprise anamino acid sequence that is at least about 70% homologous to any one ofSEQ ID NOs: 10-14. The immunoglobulin region may comprise an amino acidsequence that is at least about 80% homologous to any one of SEQ ID NOs:10-14. The immunoglobulin region may comprise an amino acid sequencethat is at least about 50% identical to any one of SEQ ID NOs: 10-14.The immunoglobulin region may comprise an amino acid sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% identical toany one of SEQ ID NOs 10-14. The immunoglobulin region may comprise anamino acid sequence that is at least about 70% identical to any one ofSEQ ID NOs: 10-14. The immunoglobulin region may comprise an amino acidsequence that is at least about 80% identical to any one of SEQ ID NOs:10-14. The immunoglobulin region may comprise an amino acid sequencethat is 100% identical to any one of SEQ ID NOs: 10-14. In someembodiments, the immunoglobulin region comprises an amino acid sequencethat is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or 97% homologous to an amino acid sequence of any one of SEQ ID NOs:10-14. In some embodiments, the immunoglobulin region comprises an aminoacid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 97% identical to an amino acid sequence of any one ofSEQ ID NOs: 10-14. The immunoglobulin region includes a Fab region thatis based on or derived from a sequence from any one of SEQ ID NOs:10-14. In some embodiments, the immunoglobulin region comprises an aminoacid Fab sequence derived from a sequence that is at least about 70%,80%, 80%, 90%, 95% or 100% to any one of SEQ ID NOs: 10-14.

The immunoglobulin region may comprise an amino acid sequence comprising10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids based on orderived from any one of SEQ ID NOs: 10-14. The immunoglobulin region maycomprise an amino acid sequence comprising 125, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 450, 500 or more aminoacids based on or derived from any one of SEQ ID NOs: 10-14. Theimmunoglobulin region may comprise an amino acid sequence comprising 10or more amino acids based on or derived from any one of SEQ ID NOs:10-14. The immunoglobulin region may comprise an amino acid sequencecomprising 50 or more amino acids based on or derived from any one ofSEQ ID NOs: 10-14. The immunoglobulin region may comprise an amino acidsequence comprising 100 or more amino acids based on or derived from anyone of SEQ ID NOs: 10-14. The immunoglobulin region may comprise anamino acid sequence comprising 200 or more amino acids based on orderived from any one of SEQ ID NOs: 10-14. The amino acids may beconsecutive. Alternatively, or additionally, the amino acids arenonconsecutive. In some embodiments, the immunoglobulin region maycomprise amino acids derived from any one of SEQ ID NOs: 10-14 and aminoacids not derived from any one of SEQ ID NOs: 10-14. In someembodiments, the immunoglobulin region may comprise amino acids derivedfrom one or more of SEQ ID NOs: 10-14 and amino acids not derived fromany one of SEQ ID NOs: 10-14. In some embodiments, the immunoglobulinregion comprises amino acids derived from 1, 2, 3, or 4 of SEQ ID NOs:10-14.

The immunoglobulin region may be encoded by a nucleotide sequence thatis based on or derived from any one of SEQ ID NOs: 3-7. Theimmunoglobulin region may be encoded by a nucleotide sequence that is atleast about 50% homologous to any one of SEQ ID NOs: 3-7. Theimmunoglobulin region may be encoded by a nucleotide sequence that is atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous toany one of SEQ ID NOs: 3-7. The immunoglobulin region may be encoded bya nucleotide sequence that is at least about 70% homologous to any oneof SEQ ID NOs: 3-7. The immunoglobulin region may be encoded by anucleotide sequence that is at least about 80% homologous to any one ofSEQ ID NOs: 3-7. The immunoglobulin region may be encoded by anucleotide sequence that is at least about 50% identical to any one ofSEQ ID NOs: 3-7. The immunoglobulin region may be encoded by anucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% identical to any one of SEQ ID NOs: 3-7. Theimmunoglobulin region may be encoded by a nucleotide sequence that is atleast about 70% identical to any one of SEQ ID NOs: 3-7. Theimmunoglobulin region may be encoded by a nucleotide sequence that is atleast about 80% identical to any one of SEQ ID NOs: 3-7. Theimmunoglobulin region may be encoded by a nucleotide sequence that is100% identical to any one of SEQ ID NOs: 3-7. The immunoglobulin regionincludes a Fab region that is based on or derived from a sequence fromany one of SEQ ID NOs: 3-7. In some embodiments, the immunoglobulinregion comprises an amino acid Fab sequence derived from a sequence thatis at least about 70%, 80%, 80%, 90%, 95% or 100% to any one of SEQ IDNOs: 3-7.

The immunoglobulin region may be encoded by a nucleotide sequencecomprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotidesbased on or derived from any one of SEQ ID NOs: 3-7. The immunoglobulinregion may be encoded by a nucleotide sequence comprising 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 450, 500 ormore nucleotides based on or derived from any one of SEQ ID NOs: 3-7.The immunoglobulin region may be encoded by a nucleotide sequencecomprising 600, 650, 700, 750, 800, 850, 900, 950, 1000 or morenucleotides based on or derived from any one of SEQ ID NOs: 3-7. Theimmunoglobulin region may be encoded by a nucleotide sequence comprising1100, 1200, 1300, 1400, 1500 or more nucleotides based on or derivedfrom any one of SEQ ID NOs: 3-7. The immunoglobulin region may beencoded by a nucleotide sequence comprising 100 or more nucleotidesbased on or derived from any one of SEQ ID NOs: 3-7. The immunoglobulinregion may be encoded by a nucleotide sequence comprising 500 or morenucleotides based on or derived from any one of SEQ ID NOs: 3-7. Theimmunoglobulin region may be encoded by a nucleotide sequence comprising1000 or more nucleotides based on or derived from any one of SEQ ID NOs:3-7. The immunoglobulin region may be encoded by a nucleotide sequencecomprising 1300 or more nucleotides based on or derived from any one ofSEQ ID NOs: 3-7. The nucleotides may be consecutive. In someembodiments, the immunoglobulin region is encoded by a nucleotidesequence comprising nucleotides derived from any one of SEQ ID NOs: 3-7and nucleotides not derived from any one of SEQ ID NOs: 3-7. In someembodiments, the immunoglobulin region is encoded by a nucleotidesequence comprising nucleotides derived from one or more of SEQ ID NOs:3-7 and nucleotides not derived from any one of SEQ ID NOs: 3-7. In someembodiments, the immunoglobulin region is encoded by a nucleotidesequence derived from 1, 2, 3, or 4 of SEQ ID NOs: 3-7.

Relaxin Therapeutic Peptide

In one aspect of the disclosure, provided herein are immunoglobulinfusion proteins comprising a therapeutic peptide and an immunoglobulinregion. The immunoglobulin fusion proteins may comprise two or moretherapeutic peptides. The immunoglobulin fusion proteins disclosedherein may comprise 3, 4, 5, or more therapeutic peptides. Thetherapeutic peptide may be attached to an immunoglobulin region via aconnecting peptide. In some embodiments, one or more additionaltherapeutic peptides are attached to the first or a secondimmunoglobulin region. The one or more therapeutic peptides may beattached to one or more immunoglobulin regions. The two or moretherapeutic peptides may be attached to two or more immunoglobulinregions. The two or more therapeutic peptides may be attached to one ormore immunoglobulin chains. The two or more therapeutic peptides may beattached to two or more immunoglobulin chains. The two or moretherapeutic peptides may be attached to one or more units within the oneor more immunoglobulin regions. The two or therapeutic peptides may beattached to two or more units within the one or more immunoglobulinregions. In some embodiments, the therapeutic peptide is connected tothe immunoglobulin region without the aid of a connecting peptide.

The immunoglobulin fusion proteins disclosed herein may comprise one ormore therapeutic agents. The therapeutic agent may be a peptide. Thetherapeutic agent may be a small molecule. The immunoglobulin fusionproteins disclosed herein may comprise two or more therapeutic agents.The immunoglobulin fusion proteins disclosed herein may comprise 3, 4,5, 6 or more therapeutic agents. The two or more therapeutic agents maybe the same. The two or more therapeutic agents may be different.

The therapeutic peptide may comprise any secondary structure, forexample alpha helix or beta strand or comprise no regular secondarystructure. The therapeutic peptide may comprise amino acids with one ormore modifications including, but not limited to, myristoylation,palmitoylation, isoprenylation, glypiation, lipoylation, acylation,acetylation, aklylation, methylation, glycosylation, malonylation,hydroxylation, iodination, nucleotide addition, oxidation,phosphorylation, adenylylation, propionylation, succinylation,sulfation, selenoylation, biotinylation, pegylation, deimination,deamidation, eliminylation, and carbamylation. The therapeutic peptidemay comprise one or more amino acids conjugated to one or more smallmolecules, for example a drug. In some embodiments, the therapeuticpeptide comprises one or more non-natural amino acids. In someembodiments, the therapeutic peptide comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or morenon-natural amino acids. In some embodiments, the therapeutic peptidecomprises one or more amino acids substitutions. In some embodiments,the therapeutic peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more amino acidsubstitutions.

In some embodiments, one or more regions of the therapeutic peptide isconfigured to treat heart failure and/or fibrosis. In some embodiments,one or more regions of the therapeutic peptide is configured to treatheart failure and/or fibrosis related conditions. In some embodiments,2, 3, 4, 5 or more regions of the therapeutic peptide are configured totreat heart failure and/or fibrosis. In some embodiments, 2, 3, 4, 5 ormore regions of the therapeutic peptide are configured to treat heartfailure and/or fibrosis related conditions. Heart failure relatedconditions may include coronary heart disease, high blood pressure,diabetes, cardiomyopathy, heart valve disease, arrhythmias, congenitalheart defects, obstructive sleep apnea, myocarditis, hyperthyroidism,hypothyroidism, emphysema, hemochromatosis, and amyloidosis. Heartfailure may be left-sided heart failure, right-sided heart failure,systolic heart failure, and diastolic heart failure. Fibrosis mayinclude, but is not limited to, pulmonary fibrosis, idiopathic pulmonaryfibrosis, cystic fibrosis, cirrhosis, endomyocardial fibrosis,myocardial infarction, atrial fibrosis, mediastinal fibrosis,myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis,nephrogenic systemic fibrosis, Crohn's disease, keloid,scleroderma/systemic sclerosis, arthrofibrosis, Peyronie's disease,Dupuytren's contracture, and adhesive capsulitis.

In some embodiments, one or more regions of the therapeutic peptidecomprises an amino acid sequence based on or derived from an amino acidsequence of a relaxin peptide. In some embodiments, one or more regionsof the therapeutic peptide comprises an amino acid sequence based on orderived from an amino acid sequence of a relaxin-2 peptide.

In some embodiments, one or more regions of the therapeutic peptide isconfigured to treat heart failure. Said heart failure may comprise oneor more of acute decompensated heart failure, right heart failure, leftheart failure, global failure, ischemic cardiomyopathy, dilatedcardiomyopathy, heart failure associated with congenital heart defects,heart failure associated with heart valve defects, mitral stenosis,mitral insufficiency, aortic stenosis, aortic insufficiency, tricuspidstenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary valveinsufficiency, heart failure associated with combined heart valvedefects, myocardial inflammation (myocarditis), chronic myocarditis,acute myocarditis, viral myocarditis, diabetic heart failure, alcoholiccardiomyopathy, heart failure associated with cardiac storage disorders,diastolic heart failure, and systolic heart failure. In someembodiments, one or more regions of the therapeutic peptide isconfigured to treat cardiovascular disease, lung disease, fibroticdisease, kidney disease, pancreatitis, inflammation, cancer,scleroderma, pulmonary fibrosis, renal fibrosis, hepatic fibrosis,fibrosis of the lung, heart, kidney, bone marrow, liver, dermatologicalfibrosis, fibrotic eye disorder, ischemia, Alzheimer's disease, cornealinjury, neurodegenerative disease, cardiovascular disease, fibroticdisease, failure, pancreatitis, inflammation, cancer, scleroderma,pulmonary fibrosis, renal fibrosis, hepatic fibrosis, thromboembolicdisorders, reperfusion damage following ischemia, micro- andmacrovascular lesions (vasculitis), arterial and venous thromboses,edemas, ischemias, myocardial infarction, stroke, transient ischemicattack, cardio protection in connection with coronary artery bypassoperations, cardio protection in connection with primary percutaneoustransluminal coronary angioplasties (PTCAs), PTCAs after thrombolysis,rescue PTCA, heart transplants and open-heart operations, organprotection in connection with transplants, bypass operations, catheterexaminations and other surgical procedures, respiratory disorders,chronic obstructive pulmonary disease, chronic bronchitis, interstitiallung disease, asthma, pulmonary emphysema, bronchiectases, cysticfibrosis (mucoviscidosis) and pulmonary hypertension, in particularpulmonary arterial hypertension, kidney disease, acute and chronickidney diseases and acute and chronic renal insufficiencies, as well asacute and chronic renal failure with or without the requirement ofdialysis, underlying or related kidney diseases, renal hypoperfusion,dialysis induced hypotension, glomerulopathies, glomerular and tubularproteinuria, renal edema, hematuria, chronic glomerulonephritis(including primary, secondary, or acute), membranous andmembranoproliferative glomerulonephritis, Alport-Syndrome,glomerulosclerosis, interstistial tubular diseases, nephropathicdiseases, primary and inborn kidney diseases, renal inflammation,immunological renal diseases, renal transplant rejection, immune complexinduced renal diseases, intoxication induced nephropathic diseases,diabetic and non-diabetic renal diseases, pyelonephritis, cystickidneys, nephrosclerosis, hypertensive nephrosclerosis, nephroticsyndrome, diseases that are characterized and diagnostically associatedwith an abnormal reduction in creatinine clearance and/or waterexcretion, abnormal increased blood concentrations of urea, nitrogen,potassium and/or creatinine, alteration in the activity of renal enzymesincluding without limitation glutamylsynthetase, urine osmolarity andurine volume, increased microalbuminuria, macroalbuminuria, glomerularand arteriolar lesions, tubular dilation, hyperphosphatemia, diseaserequiring dialysis for treatment, renal carcinomas, after incompleteresection of the kidney, dehydration after overuse of diuretics,uncontrolled blood pressure increase with malignant hypertension,urinary tract obstruction and infection, amyloidosis, systemic diseasesassociated with glomerular damage, Lupus erythematosus, rheumaticimmunological systemic diseases, renal artery stenosis, renal arterythrombosis, renal vein thrombosis, analgetics induced nephropathy,polycystic kidney disease, renal tubular acidosis, contrast mediuminduced and drug induced acute and chronic interstitial kidney diseases,metabolic syndrome, dyslipemia, aftereffects associated with acuteand/or chronic kidney diseases, pulmonary edema, heart failure, uremia,anemia, electrolyte disturbances, hyperkalemia, hyponatremia, bony andcarbohydrate metabolism, lung diseases, asthmatic disorders, pulmonaryarterial hypertension (PAH), pulmonary hypertension (PH), left-heartdisease, HIV, sickle cell anemia, thromboembolisms (CTEPH), sarcoidosis,COPD-associated pulmonary hypertension, pulmonary fibrosis-associatedpulmonary hypertension, chronic obstructive pulmonary disease (COPD),acute respiratory distress syndrome (ARDS), acute lung injury (ALI),alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, pulmonaryemphysema, pulmonary emphysema induced by cigarette smoke, cysticfibrosis (CF), fibrotic disorders, fibrotic disorders of the internalorgans, fibrotic disorders of the lung, fibrotic disorders of the heart,fibrotic disorders of the kidney, fibrotic disorders of the bone marrowfibrotic disorders of the liver, dermatological fibroses, fibrotic eyedisorders, osteodegenerative joint dysfunction, angiotensin-II(AngII)-mediated vasoconstriction, endothelin-1 (ET-1)-mediatedvasoconstriction, ischemic conditions, ischemia associated withmyocardial infarct ischemia associated with wounds, renal pathologies,renal pathologies related to vasoconstriction, hypertension,atherosclerosis, Type 1 diabetes, Type 2 diabetes, coronary arterydisease, scleroderma, stroke, diastolic dysfunction, familialhypercholesterolemia, isolated systolic hypertension, primaryhypertension, secondary hypertension, left ventricular hypertrophy,arterial stiffness associated with long-term tobacco smoking, arterialstiffness associated with obesity, arterial stiffness associated withage, systemic lupus erythematosus, preeclampsia, orhypercholesterolemia.

In some embodiments, amino acids of the therapeutic peptide, in whole orin part, are based on or derived from any one of SEQ ID NOs: 37-52. Thetherapeutic peptide may comprise an amino acid sequence that is at leastabout 50% homologous to any one of SEQ ID NOs: 37-52. The therapeuticpeptide may comprise an amino acid sequence that is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% homologous to any one of SEQID NOs: 37-52. The therapeutic peptide may comprise an amino acidsequence that is at least about 70% homologous to any one of SEQ ID NOs:37-52. The therapeutic peptide may comprise an amino acid sequence thatis at least about 80% homologous to any one of SEQ ID NOs: 37-52. Thetherapeutic peptide may comprise an amino acid sequence that is at leastabout 50% identical to any one of SEQ ID NOs: 37-52. The therapeuticpeptide may comprise an amino acid sequence that is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% identical to any one of SEQ IDNOs: 37-52. The therapeutic peptide may comprise an amino acid sequencethat is at least about 70% identical to any one of SEQ ID NOs: 37-52.The therapeutic peptide may comprise an amino acid sequence that is atleast about 80% identical to any one of SEQ ID NOs: 37-52. Thetherapeutic peptide may comprise an amino acid sequence that is 100%identical to any one of SEQ ID NOs: 37-52. In some embodiments, thetherapeutic peptide comprises an amino acid sequence that is at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97%homologous to an amino acid sequence of any one of SEQ ID NOs: 37-52. Insome embodiments, the therapeutic peptide comprises an amino acidsequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 97% identical to an amino acid sequence of any one of SEQID NOs: 37-52. In some embodiments, the therapeutic peptide comprises anamino acid sequence that is 100% identical to an amino acid sequence ofany one of SEQ ID NOs: 37-52.

The therapeutic peptide may comprise an amino acid sequence comprising10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids based on orderived from any one of SEQ ID NOs: 37-52. The therapeutic peptide maycomprise an amino acid sequence comprising 125, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 450, 500 or more aminoacids based on or derived from any one of SEQ ID NOs: 37-52. Thetherapeutic peptide may comprise an amino acid sequence comprising 10 ormore amino acids based on or derived from any one of SEQ ID NOs: 37-52.The therapeutic peptide may comprise an amino acid sequence comprising50 or more amino acids based on or derived from any one of SEQ ID NOs:37-52. The therapeutic peptide may comprise an amino acid sequencecomprising 100 or more amino acids based on or derived from any one ofSEQ ID NOs: 37-52. The therapeutic peptide may comprise an amino acidsequence comprising 200 or more amino acids based on or derived from anyone of SEQ ID NOs: 37-52. The amino acids may be consecutive.Alternatively, or additionally, the amino acids are nonconsecutive. Insome embodiments, the therapeutic peptide may comprise amino acidsderived from any one of SEQ ID NOs: 37-52 and amino acids not derivedfrom any one of SEQ ID NOs: 37-52. In some embodiments, the therapeuticpeptide may comprise amino acids derived from one or more of SEQ ID NOs:37-52 and amino acids not derived from any one of SEQ ID NOs: 37-52. Insome embodiments, the therapeutic peptide comprises amino acids derivedfrom 1, 2, 3, or 4 of SEQ ID NOs: 37-52.

The therapeutic peptide may comprise a protease cleavage site. Theprotease cleavage site may be inserted within the therapeutic peptide.In some embodiments, the therapeutic peptide comprises a firsttherapeutic peptide region and a second therapeutic peptide region. Insome embodiments, the therapeutic peptide comprises a protease cleavagesite disposed between the first therapeutic peptide region and thesecond therapeutic peptide region. In some embodiments, the firsttherapeutic peptide region and the second therapeutic peptide region arederived from the same protein or set of amino acid sequences. In someembodiments, the first therapeutic peptide region and the secondtherapeutic peptide regions are derived from different proteins or setsof amino acid sequences. The one or more protease cleavage sites may beattached to the N-terminus, C-terminus or both the N- and C-termini of aregion of a therapeutic peptide.

The therapeutic peptide may comprise one or more linker peptides. Thetherapeutic peptide may comprise two or more linker peptides. Thetherapeutic peptide may comprise 3, 4, 5, 6, 7 or more linker peptides.The linker peptides may be different. The linker peptides may be thesame. The linker peptide may be inserted within the therapeutic peptide.In some embodiments, the therapeutic peptide comprises a firsttherapeutic region, a second therapeutic region, an one or more linkerpeptides positioned between the first therapeutic region and the secondtherapeutic region. The one or more linker peptides may be attached tothe N-terminus, C-terminus or both the N- and C-termini of a region of atherapeutic peptide. In some embodiments, the linker peptide is derivedfrom amino acids of any of SEQ ID NOs: 61-62.

The therapeutic peptide may comprise one or more internal linker. Theinternal linker may be inserted within the therapeutic peptide. In someembodiments, the therapeutic peptide comprises a first therapeuticpeptide region and a second therapeutic peptide region. In someembodiments, the therapeutic peptide comprises a internal linkerdisposed between the first therapeutic peptide region and the secondtherapeutic peptide region. In some embodiments, the first therapeuticpeptide region and the second therapeutic peptide region are derivedfrom the same protein or set of amino acid sequences. In someembodiments, the first therapeutic peptide region and the secondtherapeutic peptide regions are derived from different proteins or setsof amino acid sequences. In some embodiments, the internal linker isderived from amino acids of any of SEQ ID NOs: 63-71. In someembodiments, the internal linker comprises amino acids having repeatingsequences. In some embodiments, the internal linker has 2, 3, 4, 5, 6,7, 8, 9, 10 or more repeating sequences. In some embodiments, theinternal linker is low immunogenic. In some embodiments, the internallinker is biodegradable.

Linkers and Connecting Peptides

The immunoglobulin fusion proteins, immunoglobulin regions, therapeuticpeptides, and/or non-immunoglobulin regions may further comprise one ormore linkers. The immunoglobulin fusion proteins, immunoglobulinregions, and/or non-immunoglobulin regions may further comprise 2, 3, 4,5, 6, 7, 8, 9, 10 or more linkers.

The one or more linkers are attached to the N-terminus, C-terminus orboth N- and C-termini of a therapeutic peptide. The one or more linkersare attached to the N-terminus, C-terminus or both N- and C-termini of aproteolytic cleavage site. The one or more linkers may be attached to atherapeutic peptide, proteolytic cleavage site, immunoglobulin region,non-immunoglobulin region or a combination thereof.

The one or more linkers may comprise an amino acid sequence selectedfrom any one of SEQ ID NOs:61-62. The one or more linkers may comprisean amino acid sequence that is at least about 50% homologous to any oneof SEQ ID NOs: 61-62. The one or more linkers may comprise an amino acidsequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more homologous to any one of SEQ ID NOs: 61-62. The one or morelinkers may comprise an amino acid sequence that is at least about 70%homologous to any one of SEQ ID NOs: 61-62. The one or more linkers maycomprise an amino acid sequence that is at least about 80% homologous toany one of SEQ ID NOs: 61-62.

In some embodiments, the linker is a connecting linker. The connectinglinker may link the therapeutic peptide to an immunoglobulin region. Theconnecting linker may comprise an amino acid sequence that is at leastabout 50% homologous to any of SEQ ID NOs: 53-60. The connecting linkermay comprise an amino acid sequence that is at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or more homologous to any one of SEQ IDNOs: 53-60. The connecting linker may comprise an amino acid sequencethat is at least about 70% homologous to any one of SEQ ID NOs: 53-60.The connecting linker may comprise an amino acid sequence that is atleast about 80% homologous to any one of SEQ ID NOs: 53-60.

In some embodiments, the linker is an internal linker. The internallinker may be a portion of a therapeutic peptide. The internal linkermay link two regions of a therapeutic peptide. The internal linker maylink two therapeutic peptides derived from two different peptides orproteins. The internal linker may link two therapeutic peptides derivedfrom the same peptide or protein. The internal linker may comprise anamino acid sequence that is at least about 50% homologous to any of SEQID NOs: 63-71. The internal linker may comprise an amino acid sequencethat is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or morehomologous to any one of SEQ ID NOs: 63-71. The internal linker maycomprise an amino acid sequence that is at least about 70% homologous toany one of SEQ ID NOs: 63-71. The internal linker may comprise an aminoacid sequence that is at least about 80% homologous to any one of SEQ IDNOs: 63-71.

Proteolytic Cleavage Site

The immunoglobulin fusion proteins disclosed herein may further compriseone or more proteolytic cleavage sites. The immunoglobulin fusionproteins disclosed herein may further comprise 2 or more proteolyticcleavage sites. The immunoglobulin fusion proteins disclosed herein mayfurther comprise 3 or more proteolytic cleavage sites. Theimmunoglobulin fusion proteins disclosed herein may further comprise 4,5, 6, 7 or more proteolytic cleavage sites. The therapeutic peptidesdisclosed herein may further comprise one or more proteolytic cleavagesites.

The one or more proteolytic cleavage sites may be attached to theN-terminus, C-terminus or both N- and C-termini of a therapeuticpeptide. The one or more proteolytic cleavage sites may attached to theN-terminus, C-terminus or both N- and C-termini of a linker. The one ormore proteolytic cleavage sites may be attached to a therapeuticpeptide, linker, immunoglobulin region, non-immunoglobulin region or acombination thereof.

In some embodiments, the proteolytic cleavage site is located within theamino acid sequence of the therapeutic peptide, immunoglobulin region,or a combination thereof. The therapeutic peptide may comprise one ormore proteolytic cleavage sites within its amino acid sequence. Forexample, SEQ ID NOs: 37-39 disclose a relaxin protein comprising twointernal proteolytic cleavage sites.

Two or more proteolytic cleavage sites may surround a therapeuticpeptide, linker, immunoglobulin region, or combination thereof.Digestion of the proteolytic cleavage site may result in release of apeptide fragment located between the two or more proteolytic cleavagesites. For example, the proteolytic cleavage sites may flank atherapeutic peptide-linker peptide. Digestion of the proteolyticcleavage sites may result in release of the therapeutic peptide-linker.

The proteolytic cleavage site may be recognized by one or moreproteases. The one or more proteases may be a serine protease, threonineprotease, cysteine protease, aspartate protease, glutamic protease,metalloprotease, exopeptidases, endopeptidases, or a combinationthereof. The proteases may be selected from the group comprising FactorVII or Factor Xa. Additional examples of proteases include, but are notlimited to, aminopeptidases, carboxypeptidases, trypsin, chymotrypsin,pepsin, papain, and elastase. The protease may be PC2. In someembodiments, the protease recognizes the amino acid sequence KR. In someembodiments, the protease recognizes the amino acid sequence RKKR.

Vectors, Host Cells and Recombinant Methods

Immunoglobulin fusion proteins, as disclosed herein, may be expressedand purified by known recombinant and protein purification methods. Insome instances, the activity of the immunoglobulin fusion protein isaffected by expression and/or purification methods. For example, theactivity of an immunoglobulin fusion protein configured for use as atherapeutic, is enhanced or attenuated based on the identity of theexpression vector, identity of the recombinant host, identity of thecell line, expression reaction conditions, purification methods, proteinprocessing, or any combination thereof. Expression reaction conditionsinclude, but are not limited to, temperature, % CO₂, media, expressiontime, cofactors, and chaperones. Purification methods include, but arenot limited to, purification temperatures, chromatography resins,protease inhibitors, and buffer compositions.

Immunoglobulin fusion proteins, as disclosed herein, may be expressed byrecombinant methods. Generally, a nucleic acid encoding animmunoglobulin fusion protein may be isolated and inserted into areplicable vector for further cloning (amplification of the DNA) or forexpression. DNA encoding the immunoglobulin fusion protein may beprepared by PCR amplification and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to nucleotides encoding Immunoglobulin fusionproteins). In an exemplary embodiment, nucleic acid encoding animmunoglobulin fusion protein is PCR amplified, restriction enzymedigested and gel purified. The digested nucleic acid may be insertedinto a replicable vector. The replicable vector containing the digestedimmunoglobulin fusion protein insertion may be transformed or transducedinto a host cell for further cloning (amplification of the DNA) or forexpression. Host cells may be prokaryotic or eukaryotic cells.

Polynucleotide sequences encoding polypeptide components (e.g.,immunoglobulin region, therapeutic peptide) of the immunoglobulin fusionproteins may be obtained by PCR amplification. Polynucleotide sequencesmay be isolated and sequenced from cells containing nucleic acidsencoding the polypeptide components. Alternatively, or additionally,polynucleotides may be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptide componentsmay be inserted into a recombinant vector capable of replicating andexpressing heterologous polynucleotides in prokaryotic and/or eukaryotichosts.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism may be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which may be used to transform susceptible host cells such as E.coli LE392.

Immunoglobulin fusion proteins may be expressed intracellularly (e.g.,cytoplasm) or extracellularly (e.g., secretion). For extracellularexpression, the vector may comprise a secretion signal which enablestranslocation of the immunoglobulin fusion proteins to the outside ofthe cell.

Suitable host cells for cloning or expression of immunoglobulin fusionproteins-encoding vectors include prokaryotic or eukaryotic cells. Thehost cell may be a eukaryotic. Examples of eukaryotic cells include, butare not limited to, Human Embryonic Kidney (HEK) cell, Chinese HamsterOvary (CHO) cell, fungi, yeasts, invertebrate cells (e.g., plant cellsand insect cells), lymphoid cell (e.g., YO, NSO, Sp20 cell). Otherexamples of suitable mammalian host cell lines are monkey kidney CV1line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); mousesertoli cells; monkey kidney cells (CV1); African green monkey kidneycells (VERO-76); human cervical carcinoma cells (HELA); canine kidneycells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138);human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells;MRC 5 cells; and FS4 cells. The host cell may be a prokaryotic cell(e.g., E. coli).

Host cells may be transformed with vectors containing nucleotidesencoding an immunoglobulin fusion proteins. Transformed host cells maybe cultured in media. The media may be supplemented with one or moreagents for inducing promoters, selecting transformants, or amplifying orexpressing the genes encoding the desired sequences. Methods fortransforming host cells are known in the art and may includeelectroporation, calcium chloride, or polyethylene glycol/DMSO.

Alternatively, host cells may be transfected or transduced with vectorscontaining nucleotides encoding an immunoglobulin fusion proteins.Transfected or transduced host cells may be cultured in media. The mediamay be supplemented with one or more agents for inducing promoters,selecting transfected or transduced cells, or expressing genes encodingthe desired sequences.

The expressed immunoglobulin fusion proteins may be secreted into andrecovered from the periplasm of the host cells or transported into theculture media. Protein recovery from the periplasm may involvedisrupting the host cell. Disruption of the host cell may compriseosmotic shock, sonication or lysis. Centrifugation or filtration may beused to remove cell debris or whole cells. The immunoglobulin fusionproteins may be further purified, for example, by affinity resinchromatography.

Alternatively, immunoglobulin fusion proteins that are secreted into theculture media may be isolated therein. Cells may be removed from theculture and the culture supernatant being filtered and concentrated forfurther purification of the proteins produced. The expressedpolypeptides may be further isolated and identified using commonly knownmethods such as polyacrylamide gel electrophoresis (PAGE) and Westernblot assay.

Immunoglobulin fusion proteins production may be conducted in largequantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (a preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described herein. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the immunoglobulin fusionproteins disclosed herein, various fermentation conditions may bemodified. For example, to improve the proper assembly and folding of thesecreted immunoglobulin fusion proteins polypeptides, additional vectorsoverexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB,DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-isomerasewith chaperone activity) may be used to co-transform the hostprokaryotic cells. The chaperone proteins have been demonstrated tofacilitate the proper folding and solubility of heterologous proteinsproduced in bacterial host cells.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes may be used for the presentdisclosure. For example, host cell strains may be modified to effectgenetic mutation(s) in the genes encoding known bacterial proteases suchas Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available.

Standard protein purification methods known in the art may be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography and gelfiltration using, for example, Sephadex G-75.

Immunoglobulin fusion proteins may be concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon® ultrafiltration unit.

Protease inhibitors or protease inhibitor cocktails may be included inany of the foregoing steps to inhibit proteolysis of the immunoglobulinfusion proteins.

In some cases, an immunoglobulin fusion protein may not be biologicallyactive upon isolation. Various methods for “refolding” or converting apolypeptide to its tertiary structure and generating disulfide linkages,may be used to restore biological activity. Such methods includeexposing the solubilized polypeptide to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization, but usually the chaotrope is used at a lowerconcentration and is not necessarily the same as chaotropes used for thesolubilization. In most cases the refolding/oxidation solution will alsocontain a reducing agent or the reducing agent plus its oxidized form ina specific ratio to generate a particular redox potential allowing fordisulfide shuffling to occur in the formation of the protein's cysteinebridge(s). Some of the commonly used redox couples includecysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol (DTT)/dithiane DTT, and 2-mercaptoethanol (bME)/di-thio-b(ME). In many instances, a cosolvent may be used to increase theefficiency of the refolding, and common reagents used for this purposeinclude glycerol, polyethylene glycol of various molecular weights,arginine and the like.

Compositions

Disclosed herein are compositions comprising an immunoglobulin fusionprotein and/or component of an immunoglobulin fusion protein disclosedherein. The compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore immunoglobulin fusion proteins. The immunoglobulin fusion proteinsmay be different. Alternatively, the immunoglobulin fusion proteins maybe the same or similar. The immunoglobulin fusion proteins may comprisedifferent immunoglobulin regions, therapeutic peptides or a combinationthereof.

The compositions may further comprise one or more pharmaceuticallyacceptable salts, excipients or vehicles. Pharmaceutically acceptablesalts, excipients, or vehicles for use in the present pharmaceuticalcompositions include carriers, excipients, diluents, antioxidants,preservatives, coloring, flavoring and diluting agents, emulsifyingagents, suspending agents, solvents, fillers, bulking agents, buffers,delivery vehicles, tonicity agents, cosolvents, wetting agents,complexing agents, buffering agents, antimicrobials, and surfactants.

Neutral buffered saline or saline mixed with serum albumin are exemplaryappropriate carriers. The pharmaceutical compositions may includeantioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, pluronics, or polyethylene glycol (PEG). Alsoby way of example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol, and the like. Suitable preservatives include benzalkoniumchloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid and the like. Hydrogen peroxide also may beused as preservative. Suitable cosolvents include glycerin, propyleneglycol, and PEG. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal, and the like. Thebuffers may be conventional buffers such as acetate, borate, citrate,phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH4-5.5, and Tris buffer may be about pH 7-8.5. Additional pharmaceuticalagents are set forth in Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

The composition may be in liquid form or in a lyophilized orfreeze-dried form and may include one or more lyoprotectants,excipients, surfactants, high molecular weight structural additivesand/or bulking agents (see, for example, U.S. Pat. Nos. 6,685,940,6,566,329, and 6,372,716). In one embodiment, a lyoprotectant isincluded, which is a non-reducing sugar such as sucrose, lactose ortrehalose. The amount of lyoprotectant generally included is such that,upon reconstitution, the resulting formulation will be isotonic,although hypertonic or slightly hypotonic formulations also may besuitable. In addition, the amount of lyoprotectant should be sufficientto prevent an unacceptable amount of degradation and/or aggregation ofthe protein upon lyophilization. Exemplary lyoprotectant concentrationsfor sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilizedformulation are from about 10 mM to about 400 mM. In another embodiment,a surfactant is included, such as for example, nonionic surfactants andionic surfactants such as polysorbates (e.g., polysorbate 20,polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol)phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodiumlaurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-,or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., Pluronics, PF68 etc). Exemplaryamounts of surfactant that may be present in the pre-lyophilizedformulation are from about 0.001-0.5%. High molecular weight structuraladditives (e.g., fillers, binders) may include for example, acacia,albumin, alginic acid, calcium phosphate (dibasic), cellulose,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,dextrin, dextrates, sucrose, tylose, pregelatinized starch, calciumsulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose,disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite,polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose,compressible sugar, magnesium aluminum silicate, maltodextrin,polyethylene oxide, polymethacrylates, povidone, sodium alginate,tragacanth microcrystalline cellulose, starch, and zein. Exemplaryconcentrations of high molecular weight structural additives are from0.1% to 10% by weight. In other embodiments, a bulking agent (e.g.,mannitol, glycine) may be included.

Compositions may be suitable for parenteral administration. Exemplarycompositions are suitable for injection or infusion into an animal byany route available to the skilled worker, such as intraarticular,subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, or intralesional routes. A parenteral formulationtypically will be a sterile, pyrogen-free, isotonic aqueous solution,optionally containing pharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringers'dextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, anti-microbials, antioxidants, chelating agents, inertgases and the like. See generally, Remington's Pharmaceutical Science,16th Ed., Mack Eds., 1980.

Compositions described herein may be formulated for controlled orsustained delivery in a manner that provides local concentration of theproduct (e.g., bolus, depot effect) and/or increased stability orhalf-life in a particular local environment. The compositions maycomprise the formulation of immunoglobulin fusion proteins,polypeptides, nucleic acids, or vectors disclosed herein withparticulate preparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc., as well as agents such as a biodegradablematrix, injectable microspheres, microcapsular particles, microcapsules,bioerodible particles beads, liposomes, and implantable delivery devicesthat provide for the controlled or sustained release of the active agentwhich then may be delivered as a depot injection. Techniques forformulating such sustained- or controlled-delivery means are known and avariety of polymers have been developed and used for the controlledrelease and delivery of drugs. Such polymers are typically biodegradableand biocompatible. Polymer hydrogels, including those formed bycomplexation of enantiomeric polymer or polypeptide segments, andhydrogels with temperature or pH sensitive properties, may be desirablefor providing drug depot effect because of the mild and aqueousconditions involved in trapping bioactive protein agents. See, forexample, the description of controlled release porous polymericmicroparticles for the delivery of pharmaceutical compositions in WO93/15722.

Suitable materials for this purpose include polylactides (see, e.g.,U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids),such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al.,J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyricacid. Other biodegradable polymers include poly(lactones),poly(acetals), poly(orthoesters), and poly(orthocarbonates).Sustained-release compositions also may include liposomes, which may beprepared by any of several methods known in the art (see, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrieritself, or its degradation products, should be nontoxic in the targettissue and should not further aggravate the condition. This may bedetermined by routine screening in animal models of the target disorderor, if such models are unavailable, in normal animals.

The immunoglobulin fusion proteins disclosed herein may bemicroencapsulated.

A pharmaceutical composition disclosed herein can be administered to asubject by any suitable administration route, including but not limitedto, parenteral (intravenous, subcutaneous, intraperitoneal,intramuscular, intravascular, intrathecal, intravitreal, infusion, orlocal), topical, oral, or nasal administration.

Formulations suitable for intramuscular, subcutaneous, peritumoral, orintravenous injection can include physiologically acceptable sterileaqueous or non-aqueous solutions, dispersions, suspensions or emulsions,and sterile powders for reconstitution into sterile injectable solutionsor dispersions. Examples of suitable aqueous and non-aqueous carriers,diluents, solvents, or vehicles including water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, cremophor and thelike), suitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity ismaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection also contain optional additives such aspreserving, wetting, emulsifying, and dispensing agents.

For intravenous injections, an active agent can be optionally formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer.

Parenteral injections optionally involve bolus injection or continuousinfusion. Formulations for injection are optionally presented in unitdosage form, e.g., in ampoules or in multi dose containers, with anadded preservative. The pharmaceutical composition described herein canbe in a form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of an active agent in water soluble form.Additionally, suspensions are optionally prepared as appropriate oilyinjection suspensions.

Alternatively or additionally, the compositions may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which an immunoglobulin fusionprotein disclosed herein has been absorbed or encapsulated. Where animplantation device is used, the device may be implanted into anysuitable tissue or organ, and delivery of an immunoglobulin fusionprotein, nucleic acid, or vector disclosed herein may be directlythrough the device via bolus, or via continuous administration, or viacatheter using continuous infusion.

A pharmaceutical composition comprising an immunoglobulin fusion proteindisclosed herein may be formulated for inhalation, such as for example,as a dry powder. Inhalation solutions also may be formulated in aliquefied propellant for aerosol delivery. In yet another formulation,solutions may be nebulized. Additional pharmaceutical composition forpulmonary administration include, those described, for example, in WO94/20069, which discloses pulmonary delivery of chemically modifiedproteins. For pulmonary delivery, the particle size should be suitablefor delivery to the distal lung. For example, the particle size may befrom 1 μm to 5 μm; however, larger particles may be used, for example,if each particle is fairly porous.

Certain formulations comprising an immunoglobulin fusion proteindisclosed herein may be administered orally. Formulations administeredin this fashion may be formulated with or without those carrierscustomarily used in the compounding of solid dosage forms such astablets and capsules. For example, a capsule may be designed to releasethe active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents may be includedto facilitate absorption of a selective binding agent. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders also may beemployed.

Another preparation may involve an effective quantity of animmunoglobulin fusion protein in a mixture with non-toxic excipientswhich are suitable for the manufacture of tablets. By dissolving thetablets in sterile water, or another appropriate vehicle, solutions maybe prepared in unit dose form. Suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Suitable and/or preferred pharmaceutical formulations may be determinedin view of the present disclosure and general knowledge of formulationtechnology, depending upon the intended route of administration,delivery format, and desired dosage. Regardless of the manner ofadministration, an effective dose may be calculated according to patientbody weight, body surface area, or organ size.

Further refinement of the calculations for determining the appropriatedosage for treatment involving each of the formulations described hereinare routinely made in the art and is within the ambit of tasks routinelyperformed in the art. Appropriate dosages may be ascertained through useof appropriate dose-response data.

The compositions disclosed herein may be useful for providing prognosticor providing diagnostic information.

“Pharmaceutically acceptable” may refer to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans.

“Pharmaceutically acceptable salt” may refer to a salt of a compoundthat is pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound.

“Pharmaceutically acceptable excipient, carrier or adjuvant” may referto an excipient, carrier or adjuvant that may be administered to asubject, together with at least one immunoglobulin of the presentdisclosure, and which does not destroy the pharmacological activitythereof and is nontoxic when administered in doses sufficient to delivera therapeutic amount of the compound.

“Pharmaceutically acceptable vehicle” may refer to a diluent, adjuvant,excipient, or carrier with which at least one immunoglobulin of thepresent disclosure is administered.

Kits

Further disclosed herein are kits which comprise one or moreimmunoglobulin fusion proteins or components thereof. The immunoglobulinfusion proteins may be packaged in a manner which facilitates their useto practice methods of the present disclosure. For example, a kitcomprises an immunoglobulin fusion protein described herein packaged ina container with a label affixed to the container or a package insertthat describes use of the immunoglobulin fusion protein in practicingthe method. Suitable containers include, for example, bottles, vials,syringes, etc. The containers may be formed from a variety of materialssuch as glass or plastic. The container may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). The kitmay comprise a container with an immunoglobulin fusion protein containedtherein. The kit may comprise a container with (a) an immunoglobulinregion of an immunoglobulin fusion protein. The kit may further comprisea package insert indicating that the first and second compositions maybe used to treat a particular condition. Alternatively, or additionally,the kit may further comprise a second (or third) container comprising apharmaceutically-acceptable buffer (e.g., bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution). It may further comprise other materials desirablefrom a commercial and user standpoint, including, but not limited to,other buffers, diluents, filters, needles, and syringes. Theimmunoglobulin fusion protein may be packaged in a unit dosage form. Thekit may further comprise a device suitable for administering theimmunoglobulin fusion protein according to a specific route ofadministration or for practicing a screening assay. The kit may containa label that describes use of the immunoglobulin fusion proteincomposition.

The composition comprising the immunoglobulin fusion protein may beformulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to mammals, such ashumans, bovines, felines, canines, and murines. Typically, compositionsfor intravenous administration comprise solutions in sterile isotonicaqueous buffer. Where necessary, the composition may also include asolubilizing agent and/or a local anaesthetics such as lignocaine toease pain at the site of the injection. Generally, the ingredients maybe supplied either separately or mixed together in unit dosage form. Forexample, the immunoglobulin fusion protein may be supplied as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity of theimmunoglobulin fusion protein. Where the composition is to beadministered by infusion, it may be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline may be provided so that the ingredients may bemixed prior to administration.

The amount of the composition described herein which will be effectivein the treatment, inhibition and/or prevention of a disease or disorderassociated with aberrant expression and/or activity of a therapeuticpeptide may be determined by standard clinical techniques. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation mayalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro, animalmodel test systems or clinical trials.

Therapeutic Use

Further disclosed herein are immunoglobulin fusion proteins for use inmethods of treating, alleviating, inhibiting and/or preventing one ormore diseases and/or conditions. The method may comprise administeringto a subject in need thereof a composition comprising one or moreimmunoglobulin fusion proteins disclosed herein. The immunoglobulinfusion protein may comprise an immunoglobulin region attached to atherapeutic peptide. In some embodiments, the therapeutic peptide isattached the amino terminus of the immunoglobulin region. Theimmunoglobulin fusion protein may comprise an immunoglobulin regionattached to a non-immunoglobulin region. The composition may furthercomprise a pharmaceutically acceptable carrier. The subject may be amammal. The mammal may be a human. Alternatively, the mammal is abovine. The therapeutic peptide may be a peptide or derivative orvariant thereof. Alternatively, therapeutic peptide is a small molecule.The therapeutic peptide may be relaxin or derivative or variant thereof.The immunoglobulin region may comprise one or more immunoglobulindomains. The immunoglobulin region may be an immunoglobulin A, animmunoglobulin D, an immunoglobulin E, an immunoglobulin G, or animmunoglobulin M. The immunoglobulin region may be an immunoglobulinheavy chain region or fragment thereof. In some instances, theimmunoglobulin region is from a mammalian immunoglobulin. Alternatively,the immunoglobulin region is from a chimeric immunoglobulin. Theimmunoglobulin region may be from an engineered immunoglobulin orrecombinant immunoglobulin. The immunoglobulin region may be from ahumanized, human engineered or fully human immunoglobulin. The mammalianimmunoglobulin may be a bovine immunoglobulin. The mammalianimmunoglobulin may be a human immunoglobulin. In other instances, themammalian immunoglobulin is a murine immunoglobulin. The immunoglobulinfusion protein, immunoglobulin region, and/or therapeutic peptide mayfurther comprise one or more linkers. The linker may attach aproteolytic cleavage site to the immunoglobulin region or therapeuticpeptide. The linker may be a connecting linker. The connecting linkermay connect the therapeutic peptide to the amino terminus of theimmunoglobulin region.

Further disclosed herein are immunoglobulin fusion proteins for use inmethods of treating, alleviating, inhibiting and/or preventing one ormore diseases and/or conditions. The method may comprise administeringto a subject in need thereof a composition comprising one or moreimmunoglobulin fusion proteins disclosed herein. The immunoglobulinfusion protein may comprise an immunoglobulin region comprising aPalivizumab attached to a relaxin peptide, wherein the therapeuticpeptide is attached the amino terminus of the immunoglobulin region. Theimmunoglobulin fusion protein may comprise a heavy chain fusion with anamino acid sequence selected from SEQ ID NO: 20 and 22 and a light chainwith an amino acid sequence selected from SEQ ID NO: 10-12. Theimmunoglobulin fusion protein may comprise a heavy chain fusion with anamino acid sequence at least 70% identical to SEQ ID NO: 22 and a lightchain with an amino acid sequence at least 70% identical to SEQ ID NO:12. The immunoglobulin fusion protein may comprise a heavy chain fusionwith an amino acid sequence at least 90% identical to SEQ ID NO: 22 anda light chain with an amino acid sequence at least 90% identical to SEQID NO: 12. The immunoglobulin fusion protein may comprise a heavy chainfusion with an amino acid sequence at least 95% identical to SEQ ID NO:22 and a light chain with an amino acid sequence at least 95% identicalto SEQ ID NO: 12. The immunoglobulin fusion protein may comprise a heavychain fusion with an amino acid sequence at least 99% identical to SEQID NO: 22 and a light chain with an amino acid sequence at least 99%identical to SEQ ID NO: 12. The composition may further comprise apharmaceutically acceptable carrier. The subject may be a mammal. Themammal may be a human. Alternatively, the mammal is a bovine.

The disease or condition may be an autoimmune disease, heteroimmunedisease or condition, inflammatory disease, pathogenic infection,thromboembolic disorder, respiratory disease or condition, metabolicdisease, central nervous system (CNS) disorder, bone disease or cancer.In other instances, the disease or condition is a blood disorder. Insome instances, the disease or condition is obesity, diabetes,osteoporosis, anemia, or pain. In some instances, the disease is heartrelated, for example, heart failure, acute coronary syndrome, atrialfibrillation, cardiac fibrosis, or coronary artery disease. In someembodiments, the heart failure is non-ischemic acute heart failure,chronic heart failure, acute decompensated heart failure, stablecompensated heart failure, acute heart failure, or chronic heartfailure. Additional non-limiting examples of disease and conditionsinclude, ischemia reperfusion associated with solid organ transplant(e.g., lung, kidney, liver, heart), cardiopulmonary bypass for organprotection (e.g., renal), ischemic stroke, corneal healing (ocularadministration), diabetic nephropathy, cirrhosis, portal hypertension,diabetic would healing, systemic sclerosis, cervical ripening at time oflabor, preeclampsia, portal hypertension, and fibrosis.

In some embodiments, the therapeutic peptide is exendin-4 and thedisease or condition is obesity, obesity related conditions, diabetes,and/or diabetes related conditions. In some embodiments, the therapeuticpeptide is leptin and the disease or condition is obesity, obesityrelated conditions, diabetes, and/or diabetes related conditions. Insome embodiments, the therapeutic peptide is glucagon and the disease orcondition is obesity, obesity related conditions, diabetes, and/ordiabetes related conditions. In some embodiments, the therapeuticpeptide is a glucagon analog, for example ZP1, and the disease orcondition is obesity, obesity related conditions, diabetes, and/ordiabetes related conditions. In some embodiments, the therapeuticpeptide is insulin, and the disease or condition is obesity, obesityrelated conditions, diabetes, and/or diabetes related conditions. Insome embodiments, the therapeutic peptide is oxyntomodulin, and thedisease or condition is obesity, obesity related conditions, diabetes,and/or diabetes related conditions. In some embodiments, the therapeuticpeptide is a glucagon like protein, for example GLP-1 or GLP-2, and thedisease or condition is obesity, obesity related conditions, diabetes,and/or diabetes related conditions.

In some embodiments, the therapeutic peptide is relaxin and the diseaseor condition is heart failure, heart failure related conditions,fibrosis, fibrosis related conditions, or other disease. Relaxinincludes relaxin2 and relaxins comprising internal linkers such asrelaxin2 (XT100), relaxin2 (XT35), relaxin2 (single), relaxin2 (insulinC peptide), relaxin2 (XT21), relaxin2 (30GS), relaxin2 (9GS), andrelaxin2 (GGGPRR). In some embodiments, the therapeutic peptide isrelaxin and the disease or condition is heart failure, acute coronarysyndrome, atrial fibrillation, cardiac fibrosis, or coronary arterydisease. In some embodiments, the therapeutic peptide is relaxin and thedisease or condition is ischemia reperfusion associated with solid organtransplant (e.g., lung, kidney, liver, heart), cardiopulmonary bypassfor organ protection (e.g., renal), ischemic stroke, corneal healing(ocular administration), diabetic nephropathy, cirrhosis, portalhypertension, diabetic would healing, systemic sclerosis, cervicalripening at time of labor, preeclampsia, portal hypertension, orfibrosis. In some embodiments, heart failure may comprise one or more ofacute decompensated heart failure, right heart failure, left heartfailure, global failure, ischemic cardiomyopathy, dilatedcardiomyopathy, heart failure associated with congenital heart defects,heart failure associated with heart valve defects, mitral stenosis,mitral insufficiency, aortic stenosis, aortic insufficiency, tricuspidstenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary valveinsufficiency, heart failure associated with combined heart valvedefects, myocardial inflammation (myocarditis), chronic myocarditis,acute myocarditis, viral myocarditis, diabetic heart failure, alcoholiccardiomyopathy, heart failure associated with cardiac storage disorders,diastolic heart failure, and systolic heart failure. In someembodiments, the therapeutic peptide is relaxin and the disease orcondition is cardiovascular disease, lung disease, fibrotic disease,kidney disease, pancreatitis, inflammation, cancer, scleroderma,pulmonary fibrosis, renal fibrosis, hepatic fibrosis, fibrosis of thelung, heart, kidney, bone marrow, liver, dermatological fibrosis,fibrotic eye disorder, ischemia, Alzheimer's disease, corneal injury,neurodegenerative disease, cardiovascular disease, fibrotic disease,failure, pancreatitis, inflammation, cancer, scleroderma, pulmonaryfibrosis, renal fibrosis, hepatic fibrosis, thromboembolic disorders,reperfusion damage following ischemia, micro- and macrovascular lesions(vasculitis), arterial and venous thromboses, edemas, ischemias,myocardial infarction, stroke, transient ischemic attack, cardioprotection in connection with coronary artery bypass operations, cardioprotection in connection with primary percutaneous transluminal coronaryangioplasties (PTCAs), PTCAs after thrombolysis, rescue PTCA, hearttransplants and open-heart operations, organ protection in connectionwith transplants, bypass operations, catheter examinations and othersurgical procedures, respiratory disorders, chronic obstructivepulmonary disease, chronic bronchitis, interstitial lung disease,asthma, pulmonary emphysema, bronchiectases, cystic fibrosis(mucoviscidosis) and pulmonary hypertension, in particular pulmonaryarterial hypertension, kidney disease, acute and chronic kidney diseasesand acute and chronic renal insufficiencies, as well as acute andchronic renal failure with or without the requirement of dialysis,underlying or related kidney diseases, renal hypoperfusion, dialysisinduced hypotension, glomerulopathies, glomerular and tubularproteinuria, renal edema, hematuria, chronic glomerulonephritis(including primary, secondary, or acute), membranous andmembranoproliferative glomerulonephritis, Alport-Syndrome,glomerulosclerosis, interstistial tubular diseases, nephropathicdiseases, primary and inborn kidney diseases, renal inflammation,immunological renal diseases, renal transplant rejection, immune complexinduced renal diseases, intoxication induced nephropathic diseases,diabetic and non-diabetic renal diseases, pyelonephritis, cystickidneys, nephrosclerosis, hypertensive nephrosclerosis, nephroticsyndrome, diseases that are characterized and diagnostically associatedwith an abnormal reduction in creatinine clearance and/or waterexcretion, abnormal increased blood concentrations of urea, nitrogen,potassium and/or creatinine, alteration in the activity of renal enzymesincluding without limitation glutamylsynthetase, urine osmolarity andurine volume, increased microalbuminuria, macroalbuminuria, glomerularand arteriolar lesions, tubular dilation, hyperphosphatemia, diseaserequiring dialysis for treatment, renal carcinomas, after incompleteresection of the kidney, dehydration after overuse of diuretics,uncontrolled blood pressure increase with malignant hypertension,urinary tract obstruction and infection, amyloidosis, systemic diseasesassociated with glomerular damage, Lupus erythematosus, rheumaticimmunological systemic diseases, renal artery stenosis, renal arterythrombosis, renal vein thrombosis, analgetics induced nephropathy,polycystic kidney disease, renal tubular acidosis, contrast mediuminduced and drug induced acute and chronic interstitial kidney diseases,metabolic syndrome, dyslipemia, aftereffects associated with acuteand/or chronic kidney diseases, pulmonary edema, heart failure, uremia,anemia, electrolyte disturbances, hyperkalemia, hyponatremia, bony andcarbohydrate metabolism, lung diseases, asthmatic disorders, pulmonaryarterial hypertension (PAH), pulmonary hypertension (PH), left-heartdisease, HIV, sickle cell anemia, thromboembolisms (CTEPH), sarcoidosis,COPD-associated pulmonary hypertension, pulmonary fibrosis-associatedpulmonary hypertension, chronic obstructive pulmonary disease (COPD),acute respiratory distress syndrome (ARDS), acute lung injury (ALI),alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, pulmonaryemphysema, pulmonary emphysema induced by cigarette smoke, cysticfibrosis (CF), fibrotic disorders, fibrotic disorders of the internalorgans, fibrotic disorders of the lung, fibrotic disorders of the heart,fibrotic disorders of the kidney, fibrotic disorders of the bone marrowfibrotic disorders of the liver, dermatological fibroses, fibrotic eyedisorders, osteodegenerative joint dysfunction, angiotensin-II(AngII)-mediated vasoconstriction, endothelin-1 (ET-1)-mediatedvasoconstriction, ischemic conditions, ischemia associated withmyocardial infarct ischemia associated with wounds, renal pathologies,renal pathologies related to vasoconstriction, hypertension,atherosclerosis, Type 1 diabetes, Type 2 diabetes, coronary arterydisease, scleroderma, stroke, diastolic dysfunction, familialhypercholesterolemia, isolated systolic hypertension, primaryhypertension, secondary hypertension, left ventricular hypertrophy,arterial stiffness associated with long-term tobacco smoking, arterialstiffness associated with obesity, arterial stiffness associated withage, systemic lupus erythematosus, preeclampsia, orhypercholesterolemia.

The disease and/or condition may be a chronic disease or condition.Alternatively, the disease and/or condition is an acute disease orcondition. The disease or condition may be recurrent, refractory,accelerated, or in remission. The disease or condition may affect one ormore cell types. The one or more diseases and/or conditions may be anautoimmune disease, inflammatory disease, cardiovascular disease,metabolic disorder, pregnancy, and cell proliferative disorder.

The disease or condition may be an autoimmune disease. In some cases,the autoimmune disease may be scleroderma, diffuse scleroderma orsystemic scleroderma.

The disease or condition may be an inflammatory disease. In some cases,the inflammatory disease may be hepatitis, fibromyalgia or psoriasis.

The disease or condition may be a rheumatic disease. In some cases, therheumatic disease may be Ankylosing spondylitis, back pain, bursitis,tendinitis, shoulder pain, wrist pain, bicep pain, leg pain, knee pain,ankle pain, hip pain, Achilles pain, Capsulitis, neck pain,osteoarthritis, systemic lupus, erythematosus, rheumatoid arthritis,juvenile arthritis, Sjögren syndrome, Polymyositis, Behçet's disease,Reiter's syndrome, or Psoriatic arthritis. The rheumatic disease may bechronic. Alternatively, the rheumatic disease is acute.

The disease or condition may be a cardiovascular disease. In some cases,the cardiovascular disease may be acute heart failure, congestive heartfailure, compensated heart failure, decompensated heart failure,hypercholesterolemia, atherosclerosis, coronary heart disease orischemic stroke. The cardiovascular disease may be cardiac hypertrophy.

The disease or condition may be a metabolic disorder. In some cases, themetabolic disorder may be hypercholesterolemia, hypobetalipoproteinemia,hypertriglyceridemia, hyperlipidemia, dyslipidemia, ketosis,hypolipidemia, refractory anemia, appetite control, gastric emptying,non-alcoholic fatty liver disease, obesity, type I diabetes mellitus,type II diabetes mellitus, gestational diabetes mellitus, metabolicsyndrome. The metabolic disorder may be type I diabetes. The metabolicdisorder may be type II diabetes.

The disease or condition may be pregnancy. The immunoglobulin fusionproteins may be used to treat preeclampsia or induce labor.

Disclosed herein are methods of treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject a composition comprising an immunoglobulin fusion proteindisclosed herein. In some embodiments, the immunoglobulin fusion proteincomprises a therapeutic peptide attached to an immunoglobulin region. Insome embodiments, the therapeutic peptide is attached to theimmunoglobulin region via a chemical linker referred to as a connectingpeptide. In some embodiments, the therapeutic peptide is attached to theamino terminus of the immunoglobulin region. In some embodiments, thetherapeutic peptide is relaxin. In some embodiments, the immunoglobulinfusion protein comprises an immunoglobulin region comprising aPalivizumab attached to a relaxin peptide, wherein the therapeuticpeptide is attached the amino terminus of the immunoglobulin region. Insome embodiments, the immunoglobulin fusion protein comprises a heavychain fusion with an amino acid sequence selected from SEQ ID NO: 20 and22 and a light chain with an amino acid sequence selected from SEQ IDNO: 10-12. In some embodiments, the immunoglobulin fusion proteincomprises a heavy chain fusion with an amino acid sequence at least 70%identical to SEQ ID NO: 22 and a light chain with an amino acid sequenceat least 70% identical to SEQ ID NO: 12. In some embodiments, theimmunoglobulin fusion protein comprises a heavy chain fusion with anamino acid sequence at least 90% identical to SEQ ID NO: 22 and a lightchain with an amino acid sequence at least 90% identical to SEQ ID NO:12. In some embodiments, the immunoglobulin fusion protein comprises aheavy chain fusion with an amino acid sequence at least 95% identical toSEQ ID NO: 22 and a light chain with an amino acid sequence at least 95%identical to SEQ ID NO: 12. In some embodiments, the immunoglobulinfusion protein comprises a heavy chain fusion with an amino acidsequence at least 99% identical to SEQ ID NO: 22 and a light chain withan amino acid sequence at least 99% identical to SEQ ID NO: 12. Thedisease or condition may be a cardiovascular disease. The cardiovasculardisease may be acute heart failure. Additional cardiovascular diseasesinclude, but are not limited to, congestive heart failure, compensatedheart failure or decompensated heart failure. The disease or conditionmay be an autoimmune disorder. The autoimmune disorder may bescleroderma, diffuse scleroderma or systemic scleroderma. The disease orcondition may be an inflammatory disease. The inflammatory disease maybe fibromyalgia. The disease or condition may be fibrosis.Alternatively, the disease or condition is pregnancy. The immunoglobulinfusion protein may be used to treat preeclampsia or induce labor. Insome embodiments, heart failure may comprise one or more of acutedecompensated heart failure, right heart failure, left heart failure,global failure, ischemic cardiomyopathy, dilated cardiomyopathy, heartfailure associated with congenital heart defects, heart failureassociated with heart valve defects, mitral stenosis, mitralinsufficiency, aortic stenosis, aortic insufficiency, tricuspidstenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary valveinsufficiency, heart failure associated with combined heart valvedefects, myocardial inflammation (myocarditis), chronic myocarditis,acute myocarditis, viral myocarditis, diabetic heart failure, alcoholiccardiomyopathy, heart failure associated with cardiac storage disorders,diastolic heart failure, and systolic heart failure. In someembodiments, the immunoglobulin fusion protein may be used to treat adisease selected from the group consisting of cardiovascular disease,lung disease, fibrotic disease, kidney disease, pancreatitis,inflammation, cancer, scleroderma, pulmonary fibrosis, renal fibrosis,hepatic fibrosis, fibrosis of the lung, heart, kidney, bone marrow,liver, dermatological fibrosis, fibrotic eye disorder, ischemia,Alzheimer's disease, corneal injury, neurodegenerative disease,cardiovascular disease, fibrotic disease, failure, pancreatitis,inflammation, cancer, scleroderma, pulmonary fibrosis, renal fibrosis,hepatic fibrosis, thromboembolic disorders, reperfusion damage followingischemia, micro- and macrovascular lesions (vasculitis), arterial andvenous thromboses, edemas, ischemias, myocardial infarction, stroke,transient ischemic attack, cardio protection in connection with coronaryartery bypass operations, cardio protection in connection with primarypercutaneous transluminal coronary angioplasties (PTCAs), PTCAs afterthrombolysis, rescue PTCA, heart transplants and open-heart operations,organ protection in connection with transplants, bypass operations,catheter examinations and other surgical procedures, respiratorydisorders, chronic obstructive pulmonary disease, chronic bronchitis,interstitial lung disease, asthma, pulmonary emphysema, bronchiectases,cystic fibrosis (mucoviscidosis) and pulmonary hypertension, inparticular pulmonary arterial hypertension, kidney disease, acute andchronic kidney diseases and acute and chronic renal insufficiencies, aswell as acute and chronic renal failure with or without the requirementof dialysis, underlying or related kidney diseases, renal hypoperfusion,dialysis induced hypotension, glomerulopathies, glomerular and tubularproteinuria, renal edema, hematuria, chronic glomerulonephritis(including primary, secondary, or acute), membranous andmembranoproliferative glomerulonephritis, Alport-Syndrome,glomerulosclerosis, interstistial tubular diseases, nephropathicdiseases, primary and inborn kidney diseases, renal inflammation,immunological renal diseases, renal transplant rejection, immune complexinduced renal diseases, intoxication induced nephropathic diseases,diabetic and non-diabetic renal diseases, pyelonephritis, cystickidneys, nephrosclerosis, hypertensive nephrosclerosis, nephroticsyndrome, diseases that are characterized and diagnostically associatedwith an abnormal reduction in creatinine clearance and/or waterexcretion, abnormal increased blood concentrations of urea, nitrogen,potassium and/or creatinine, alteration in the activity of renal enzymesincluding without limitation glutamylsynthetase, urine osmolarity andurine volume, increased microalbuminuria, macroalbuminuria, glomerularand arteriolar lesions, tubular dilation, hyperphosphatemia, diseaserequiring dialysis for treatment, renal carcinomas, after incompleteresection of the kidney, dehydration after overuse of diuretics,uncontrolled blood pressure increase with malignant hypertension,urinary tract obstruction and infection, amyloidosis, systemic diseasesassociated with glomerular damage, Lupus erythematosus, rheumaticimmunological systemic diseases, renal artery stenosis, renal arterythrombosis, renal vein thrombosis, analgetics induced nephropathy,polycystic kidney disease, renal tubular acidosis, contrast mediuminduced and drug induced acute and chronic interstitial kidney diseases,metabolic syndrome, dyslipemia, aftereffects associated with acuteand/or chronic kidney diseases, pulmonary edema, heart failure, uremia,anemia, electrolyte disturbances, hyperkalemia, hyponatremia, bony andcarbohydrate metabolism, lung diseases, asthmatic disorders, pulmonaryarterial hypertension (PAH), pulmonary hypertension (PH), left-heartdisease, HIV, sickle cell anemia, thromboembolisms (CTEPH), sarcoidosis,COPD-associated pulmonary hypertension, pulmonary fibrosis-associatedpulmonary hypertension, chronic obstructive pulmonary disease (COPD),acute respiratory distress syndrome (ARDS), acute lung injury (ALI),alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, pulmonaryemphysema, pulmonary emphysema induced by cigarette smoke, cysticfibrosis (CF), fibrotic disorders, fibrotic disorders of the internalorgans, fibrotic disorders of the lung, fibrotic disorders of the heart,fibrotic disorders of the kidney, fibrotic disorders of the bone marrowfibrotic disorders of the liver, dermatological fibroses, fibrotic eyedisorders, osteodegenerative joint dysfunction, angiotensin-II(AngII)-mediated vasoconstriction, endothelin-1 (ET-1)-mediatedvasoconstriction, ischemic conditions, ischemia associated withmyocardial infarct ischemia associated with wounds, renal pathologies,renal pathologies related to vasoconstriction, hypertension,atherosclerosis, Type 1 diabetes, Type 2 diabetes, coronary arterydisease, scleroderma, stroke, diastolic dysfunction, familialhypercholesterolemia, isolated systolic hypertension, primaryhypertension, secondary hypertension, left ventricular hypertrophy,arterial stiffness associated with long-term tobacco smoking, arterialstiffness associated with obesity, arterial stiffness associated withage, systemic lupus erythematosus, preeclampsia, andhypercholesterolemia.

Disclosed herein may be a method of preventing or treating a disease orcondition in a subject in need thereof comprising administering to thesubject a composition comprising one or more immunoglobulin fusionproteins disclosed herein. The immunoglobulin fusion protein maycomprise an immunoglobulin region attached to therapeutic peptide. Theimmunoglobulin fusion protein may comprise one or more immunoglobulinheavy chains, light chains, or a combination thereof. The immunoglobulinfusion protein sequence may share 50%, 60%, 70%, 80%, 85%, 90%, 95%,97%, 99%, or more amino acid sequence identity to a heavy chain sequenceprovided by SEQ ID NOs: 20-22. The immunoglobulin fusion proteinsequence may share 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or moreamino acid sequence identity to a heavy chain sequence provided by SEQID NO: 22. The immunoglobulin fusion protein sequence may share 50%,60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more amino acid sequenceidentity to a light chain sequence provided by SEQ ID NOs: 9-12. Theimmunoglobulin fusion protein sequence may share 50%, 60%, 70%, 80%,85%, 90%, 95%, 97%, 99%, or more amino acid sequence identity to a lightchain sequence provided by SEQ ID NO: 12. The immunoglobulin heavy chainmay be encoded by a nucleotide sequence that is at least about 50%, 60%,70%, 80%, 85%, 90%, 95%, 97%, 99%, or more homologous to SEQ ID NOs:17-19. The immunoglobulin heavy chain may be encoded by a nucleotidesequence that is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%,99%, or more homologous to SEQ ID NO: 19. The immunoglobulin light chainmay be encoded by a nucleotide sequence that is at least about 50%, 60%,70%, 80%, 85%, 90%, 95%, 97%, 99%, or more homologous to SEQ ID NOs:1-4. The immunoglobulin light chain may be encoded by a nucleotidesequence that is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%,99%, or more homologous to SEQ ID NO: 4. The immunoglobulin fusionprotein may further comprise one or more linkers. The immunoglobulinfusion protein may further comprise one or more internal linkers. Theimmunoglobulin fusion protein may further comprise one or moreproteolytic cleavage sites. The disease or condition may be anautoimmune disease, heteroimmune disease or condition, inflammatorydisease, pathogenic infection, thromboembolic disorder, respiratorydisease or condition, metabolic disease, central nervous system (CNS)disorder, bone disease or cancer. The disease or condition may be ablood disorder. In some instances, the disease or condition may beobesity, diabetes, osteoporosis, anemia, or pain. In some embodiments,the disease or condition is heart failure, acute coronary syndrome,atrial fibrillation, cardiac fibrosis, or coronary artery disease. Insome embodiments, the disease or condition is ischemia reperfusionassociated with solid organ transplant (e.g., lung, kidney, liver,heart), cardiopulmonary bypass for organ protection (e.g., renal),ischemic stroke, corneal healing (ocular administration), diabeticnephropathy, cirrhosis, portal hypertension, diabetic would healing,systemic sclerosis, cervical ripening at time of labor, preeclampsia,portal hypertension, or fibrosis. In some embodiments, the disease orcondition may be heart failure. Said heart failure may comprise one ormore of acute decompensated heart failure, right heart failure, leftheart failure, global failure, ischemic cardiomyopathy, dilatedcardiomyopathy, heart failure associated with congenital heart defects,heart failure associated with heart valve defects, mitral stenosis,mitral insufficiency, aortic stenosis, aortic insufficiency, tricuspidstenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary valveinsufficiency, heart failure associated with combined heart valvedefects, myocardial inflammation (myocarditis), chronic myocarditis,acute myocarditis, viral myocarditis, diabetic heart failure, alcoholiccardiomyopathy, heart failure associated with cardiac storage disorders,diastolic heart failure, and systolic heart failure. In someembodiments, the disease or condition is cardiovascular disease, lungdisease, fibrotic disease, kidney disease, pancreatitis, inflammation,cancer, scleroderma, pulmonary fibrosis, renal fibrosis, hepaticfibrosis, fibrosis of the lung, heart, kidney, bone marrow, liver,dermatological fibrosis, fibrotic eye disorder, ischemia, Alzheimer'sdisease, corneal injury, neurodegenerative disease, cardiovasculardisease, fibrotic disease, failure, pancreatitis, inflammation, cancer,scleroderma, pulmonary fibrosis, renal fibrosis, hepatic fibrosis,thromboembolic disorders, reperfusion damage following ischemia, micro-and macrovascular lesions (vasculitis), arterial and venous thromboses,edemas, ischemias, myocardial infarction, stroke, transient ischemicattack, cardio protection in connection with coronary artery bypassoperations, cardio protection in connection with primary percutaneoustransluminal coronary angioplasties (PTCAs), PTCAs after thrombolysis,rescue PTCA, heart transplants and open-heart operations, organprotection in connection with transplants, bypass operations, catheterexaminations and other surgical procedures, respiratory disorders,chronic obstructive pulmonary disease, chronic bronchitis, interstitiallung disease, asthma, pulmonary emphysema, bronchiectases, cysticfibrosis (mucoviscidosis) and pulmonary hypertension, in particularpulmonary arterial hypertension, kidney disease, acute and chronickidney diseases and acute and chronic renal insufficiencies, as well asacute and chronic renal failure with or without the requirement ofdialysis, underlying or related kidney diseases, renal hypoperfusion,dialysis induced hypotension, glomerulopathies, glomerular and tubularproteinuria, renal edema, hematuria, chronic glomerulonephritis(including primary, secondary, or acute), membranous andmembranoproliferative glomerulonephritis, Alport-Syndrome,glomerulosclerosis, interstistial tubular diseases, nephropathicdiseases, primary and inborn kidney diseases, renal inflammation,immunological renal diseases, renal transplant rejection, immune complexinduced renal diseases, intoxication induced nephropathic diseases,diabetic and non-diabetic renal diseases, pyelonephritis, cystickidneys, nephrosclerosis, hypertensive nephrosclerosis, nephroticsyndrome, diseases that are characterized and diagnostically associatedwith an abnormal reduction in creatinine clearance and/or waterexcretion, abnormal increased blood concentrations of urea, nitrogen,potassium and/or creatinine, alteration in the activity of renal enzymesincluding without limitation glutamylsynthetase, urine osmolarity andurine volume, increased microalbuminuria, macroalbuminuria, glomerularand arteriolar lesions, tubular dilation, hyperphosphatemia, diseaserequiring dialysis for treatment, renal carcinomas, after incompleteresection of the kidney, dehydration after overuse of diuretics,uncontrolled blood pressure increase with malignant hypertension,urinary tract obstruction and infection, amyloidosis, systemic diseasesassociated with glomerular damage, Lupus erythematosus, rheumaticimmunological systemic diseases, renal artery stenosis, renal arterythrombosis, renal vein thrombosis, analgetics induced nephropathy,polycystic kidney disease, renal tubular acidosis, contrast mediuminduced and drug induced acute and chronic interstitial kidney diseases,metabolic syndrome, dyslipemia, aftereffects associated with acuteand/or chronic kidney diseases, pulmonary edema, heart failure, uremia,anemia, electrolyte disturbances, hyperkalemia, hyponatremia, bony andcarbohydrate metabolism, lung diseases, asthmatic disorders, pulmonaryarterial hypertension (PAH), pulmonary hypertension (PH), left-heartdisease, HIV, sickle cell anemia, thromboembolisms (CTEPH), sarcoidosis,COPD-associated pulmonary hypertension, pulmonary fibrosis-associatedpulmonary hypertension, chronic obstructive pulmonary disease (COPD),acute respiratory distress syndrome (ARDS), acute lung injury (ALI),alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, pulmonaryemphysema, pulmonary emphysema induced by cigarette smoke, cysticfibrosis (CF), fibrotic disorders, fibrotic disorders of the internalorgans, fibrotic disorders of the lung, fibrotic disorders of the heart,fibrotic disorders of the kidney, fibrotic disorders of the bone marrowfibrotic disorders of the liver, dermatological fibroses, fibrotic eyedisorders, osteodegenerative joint dysfunction, angiotensin-II(AngII)-mediated vasoconstriction, endothelin-1 (ET-1)-mediatedvasoconstriction, ischemic conditions, ischemia associated withmyocardial infarct ischemia associated with wounds, renal pathologies,renal pathologies related to vasoconstriction, hypertension,atherosclerosis, Type 1 diabetes, Type 2 diabetes, coronary arterydisease, scleroderma, stroke, diastolic dysfunction, familialhypercholesterolemia, isolated systolic hypertension, primaryhypertension, secondary hypertension, left ventricular hypertrophy,arterial stiffness associated with long-term tobacco smoking, arterialstiffness associated with obesity, arterial stiffness associated withage, systemic lupus erythematosus, preeclampsia, orhypercholesterolemia.

Disclosed herein is a method of preventing or treating heart failure ina subject in need thereof comprising administering to the subject acomposition comprising one or more immunoglobulin fusion proteinsdescribed herein, wherein the immunoglobulin fusion protein comprises aheavy chain fusion having an amino acid sequence at least 90% identicalto SEQ ID NOs: 20 or 22 and a light chain having an amino acid sequenceat least 90% identical to SEQ ID NOs: 10-12. In some embodiments, theimmunoglobulin fusion protein comprises a heavy chain fusion having anamino acid sequence at least 90% identical to SEQ ID NOs: 22 and a lightchain having an amino acid sequence at least 90% identical to SEQ IDNOs: 12. The composition may further comprise a pharmaceuticallyacceptable carrier. The subject may be a mammal. The mammal may be ahuman. The heart failure may comprise one or more of acute decompensatedheart failure, right heart failure, left heart failure, global failure,ischemic cardiomyopathy, dilated cardiomyopathy, heart failureassociated with congenital heart defects, heart failure associated withheart valve defects, mitral stenosis, mitral insufficiency, aorticstenosis, aortic insufficiency, tricuspid stenosis, tricuspidinsufficiency, pulmonary stenosis, pulmonary valve insufficiency, heartfailure associated with combined heart valve defects, myocardialinflammation (myocarditis), chronic myocarditis, acute myocarditis,viral myocarditis, diabetic heart failure, alcoholic cardiomyopathy,heart failure associated with cardiac storage disorders, diastolic heartfailure, and systolic heart failure.

Provided are methods of treatment, inhibition and prevention of adisease or condition in a subject in need thereof by administration tothe subject of an effective amount of an immunoglobulin fusion proteinor pharmaceutical composition described herein. The immunoglobulinfusion protein may be substantially purified (e.g., substantially freefrom substances that limit its effect or produce undesiredside-effects). The subject may be an animal, including but not limitedto animals such as cows, pigs, sheep, goats, rabbits, horses, chickens,cats, dogs, mice, etc. The subject may be a mammal. The subject may be ahuman. The subject may be a non-human primate. Alternatively, thesubject may be a bovine. The subject may be an avian, reptile oramphibian.

Methods of treatment disclosed herein comprise administering animmunoglobulin fusion protein comprises an immunoglobulin regioncomprising a Palivizumab attached to a relaxin peptide, wherein thetherapeutic peptide is attached the amino terminus of the immunoglobulinregion according to a schedule. In some embodiments, the immunoglobulinfusion protein is administered twice per week. In some embodiments, theimmunoglobulin fusion protein is administered once per week. In someembodiments, the immunoglobulin fusion protein is administered once pertwo weeks. In some embodiments, the immunoglobulin fusion protein isadministered four times per month. In some embodiments, theimmunoglobulin fusion protein is administered three times per month. Insome embodiments, the immunoglobulin fusion protein is administered onceper month. In some embodiments, the immunoglobulin fusion protein isadministered in an infusion lasting an indicated amount of time. In someembodiments, the immunoglobulin fusion protein is administered for sixhours. In some embodiments, the immunoglobulin fusion protein isadministered for five hours. In some embodiments, the immunoglobulinfusion protein is administered for four hours. In some embodiments, theimmunoglobulin fusion protein is administered for three hours. In someembodiments, the immunoglobulin fusion protein is administered for twohours. In some embodiments, the immunoglobulin fusion protein isadministered for one hour. In some embodiments, the immunoglobulinfusion protein is administered subcutaneously. In some embodiments, theimmunoglobulin fusion protein is administered intramuscularly. In someembodiments, the immunoglobulin fusion protein is administeredintravenously.

Additional Uses

Further disclosed herein are uses of an immunoglobulin fusion protein inthe manufacture of a medicament for the treatment of a disease orcondition. The immunoglobulin fusion protein may be any of theimmunoglobulin fusion proteins disclosed herein. Disclosed herein is theuse of an immunoglobulin fusion protein in the manufacture of amedicament for the treatment of a disease or condition, theimmunoglobulin fusion protein comprising an immunoglobulin regionattached to a therapeutic peptide. In some embodiments, the therapeuticpeptide is attached to the amino terminus of an immunoglobulin region.Further disclosed herein is the use of an immunoglobulin fusion proteinin the manufacture of a medicament for the treatment of a disease orcondition, the immunoglobulin fusion protein comprising animmunoglobulin region attached to a therapeutic peptide. In someembodiments, the therapeutic peptide is attached to the amino terminusof an immunoglobulin region. The immunoglobulin fusion protein maycomprise one or more internal linkers, one or more protease cleavagesites, one or more connecting peptides, and any combination thereof. Theone or more internal linkers, one or more protease cleavage sites,and/or one or more connecting peptides may be inserted within theimmunoglobulin region. The one or more internal linkers, one or moreprotease cleavage sites, and/or one or more connecting peptides may beinserted within the therapeutic peptide. The one or more internallinkers, one or more protease cleavage sites, and/or one or moreconnecting peptides may be connected to the amino terminus of theimmunoglobulin region. The immunoglobulin region may comprise one ormore immunoglobulin domains. The immunoglobulin domain may be animmunoglobulin A, an immunoglobulin D, an immunoglobulin E, animmunoglobulin G, or an immunoglobulin M. The immunoglobulin domain maybe an immunoglobulin heavy chain region or fragment thereof. Theimmunoglobulin domain may be an immunoglobulin light chain region orfragment thereof. The immunoglobulin domain may be from an anti-viral,anti-bacterial, anti-parasitic, and/or anti-fungal immunoglobulin. Insome instances, the immunoglobulin domain is from a mammalianimmunoglobulin. Alternatively, the immunoglobulin domain is from achimeric immunoglobulin. The immunoglobulin domain may be from anengineered immunoglobulin or recombinant immunoglobulin. Theimmunoglobulin domain may be from a humanized, human engineered or fullyhuman immunoglobulin. The mammalian immunoglobulin may be a bovineimmunoglobulin. The mammalian immunoglobulin may be a humanimmunoglobulin. In other instances, the mammalian immunoglobulin is amurine immunoglobulin. The therapeutic peptide may be a peptide orderivative or variant thereof. The therapeutic peptide may compriserelaxin. The disease or condition may be an autoimmune disease,heteroimmune disease or condition, inflammatory disease, pathogenicinfection, thromboembolic disorder, respiratory disease or condition,metabolic disease, central nervous system (CNS) disorder, bone diseaseor cancer. In other instances, the disease or condition is a blooddisorder. In some instances, the disease or condition is obesity,diabetes, osteoporosis, anemia, or pain. The disease or condition may bea growth disorder. In some embodiments, the disease or condition isheart failure, acute coronary syndrome, atrial fibrillation, cardiacfibrosis, or coronary artery disease. In some embodiments, the diseaseor condition is ischemia reperfusion associated with solid organtransplant (e.g., lung, kidney, liver, heart), cardiopulmonary bypassfor organ protection (e.g., renal), ischemic stroke, corneal healing(ocular administration), diabetic nephropathy, cirrhosis, portalhypertension, diabetic would healing, systemic sclerosis, cervicalripening at time of labor, preeclampsia, portal hypertension, orfibrosis.

Disclosed herein are uses of an immunoglobulin fusion protein in themanufacture of a medicament for the treatment of a disease or condition.In some embodiments, the immunoglobulin fusion protein comprises atherapeutic peptide attached to an immunoglobulin region. In someembodiments, the therapeutic peptide is attached to the immunoglobulinregion via a chemical linker referred to as a connecting peptide. Insome embodiments, the therapeutic peptide is attached to the aminoterminus of the immunoglobulin region. In some embodiments, thetherapeutic peptide is relaxin. In some embodiments, the immunoglobulinfusion protein comprises an immunoglobulin region comprising aPalivizumab attached to a relaxin peptide, wherein the therapeuticpeptide is attached the amino terminus of the immunoglobulin region. Insome embodiments, the immunoglobulin fusion protein comprises a heavychain fusion with an amino acid sequence selected from SEQ ID NO: 20 and22 and a light chain with an amino acid sequence selected from SEQ IDNO: 10-12. In some embodiments, the immunoglobulin fusion proteincomprises a heavy chain fusion with an amino acid sequence at least 70%identical to SEQ ID NO: 22 and a light chain with an amino acid sequenceat least 70% identical to SEQ ID NO: 12. In some embodiments, theimmunoglobulin fusion protein comprises a heavy chain fusion with anamino acid sequence at least 90% identical to SEQ ID NO: 22 and a lightchain with an amino acid sequence at least 90% identical to SEQ ID NO:12. In some embodiments, the immunoglobulin fusion protein comprises aheavy chain fusion with an amino acid sequence at least 95% identical toSEQ ID NO: 22 and a light chain with an amino acid sequence at least 95%identical to SEQ ID NO: 12. In some embodiments, the immunoglobulinfusion protein comprises a heavy chain fusion with an amino acidsequence at least 99% identical to SEQ ID NO: 22 and a light chain withan amino acid sequence at least 99% identical to SEQ ID NO: 12. Thedisease or condition may be a cardiovascular disease. The cardiovasculardisease may be acute heart failure. Additional cardiovascular diseasesinclude, but are not limited to, congestive heart failure, compensatedheart failure or decompensated heart failure. The disease or conditionmay be an autoimmune disorder. The autoimmune disorder may bescleroderma, diffuse scleroderma or systemic scleroderma. The disease orcondition may be an inflammatory disease. The inflammatory disease maybe fibromyalgia. The disease or condition may be fibrosis.Alternatively, the disease or condition is pregnancy. The immunoglobulinfusion protein may be used to treat preeclampsia or induce labor. Insome embodiments, heart failure may comprise one or more of acutedecompensated heart failure, right heart failure, left heart failure,global failure, ischemic cardiomyopathy, dilated cardiomyopathy, heartfailure associated with congenital heart defects, heart failureassociated with heart valve defects, mitral stenosis, mitralinsufficiency, aortic stenosis, aortic insufficiency, tricuspidstenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary valveinsufficiency, heart failure associated with combined heart valvedefects, myocardial inflammation (myocarditis), chronic myocarditis,acute myocarditis, viral myocarditis, diabetic heart failure, alcoholiccardiomyopathy, heart failure associated with cardiac storage disorders,diastolic heart failure, and systolic heart failure. In someembodiments, the immunoglobulin fusion protein may be used to treat adisease selected from the group consisting of cardiovascular disease,lung disease, fibrotic disease, kidney disease, pancreatitis,inflammation, cancer, scleroderma, pulmonary fibrosis, renal fibrosis,hepatic fibrosis, fibrosis of the lung, heart, kidney, bone marrow,liver, dermatological fibrosis, fibrotic eye disorder, ischemia,Alzheimer's disease, corneal injury, neurodegenerative disease,cardiovascular disease, fibrotic disease, failure, pancreatitis,inflammation, cancer, scleroderma, pulmonary fibrosis, renal fibrosis,hepatic fibrosis, thromboembolic disorders, reperfusion damage followingischemia, micro- and macrovascular lesions (vasculitis), arterial andvenous thromboses, edemas, ischemias, myocardial infarction, stroke,transient ischemic attack, cardio protection in connection with coronaryartery bypass operations, cardio protection in connection with primarypercutaneous transluminal coronary angioplasties (PTCAs), PTCAs afterthrombolysis, rescue PTCA, heart transplants and open-heart operations,organ protection in connection with transplants, bypass operations,catheter examinations and other surgical procedures, respiratorydisorders, chronic obstructive pulmonary disease, chronic bronchitis,interstitial lung disease, asthma, pulmonary emphysema, bronchiectases,cystic fibrosis (mucoviscidosis) and pulmonary hypertension, inparticular pulmonary arterial hypertension, kidney disease, acute andchronic kidney diseases and acute and chronic renal insufficiencies, aswell as acute and chronic renal failure with or without the requirementof dialysis, underlying or related kidney diseases, renal hypoperfusion,dialysis induced hypotension, glomerulopathies, glomerular and tubularproteinuria, renal edema, hematuria, chronic glomerulonephritis(including primary, secondary, or acute), membranous andmembranoproliferative glomerulonephritis, Alport-Syndrome,glomerulosclerosis, interstistial tubular diseases, nephropathicdiseases, primary and inborn kidney diseases, renal inflammation,immunological renal diseases, renal transplant rejection, immune complexinduced renal diseases, intoxication induced nephropathic diseases,diabetic and non-diabetic renal diseases, pyelonephritis, cystickidneys, nephrosclerosis, hypertensive nephrosclerosis, nephroticsyndrome, diseases that are characterized and diagnostically associatedwith an abnormal reduction in creatinine clearance and/or waterexcretion, abnormal increased blood concentrations of urea, nitrogen,potassium and/or creatinine, alteration in the activity of renal enzymesincluding without limitation glutamylsynthetase, urine osmolarity andurine volume, increased microalbuminuria, macroalbuminuria, glomerularand arteriolar lesions, tubular dilation, hyperphosphatemia, diseaserequiring dialysis for treatment, renal carcinomas, after incompleteresection of the kidney, dehydration after overuse of diuretics,uncontrolled blood pressure increase with malignant hypertension,urinary tract obstruction and infection, amyloidosis, systemic diseasesassociated with glomerular damage, Lupus erythematosus, rheumaticimmunological systemic diseases, renal artery stenosis, renal arterythrombosis, renal vein thrombosis, analgetics induced nephropathy,polycystic kidney disease, renal tubular acidosis, contrast mediuminduced and drug induced acute and chronic interstitial kidney diseases,metabolic syndrome, dyslipemia, aftereffects associated with acuteand/or chronic kidney diseases, pulmonary edema, heart failure, uremia,anemia, electrolyte disturbances, hyperkalemia, hyponatremia, bony andcarbohydrate metabolism, lung diseases, asthmatic disorders, pulmonaryarterial hypertension (PAH), pulmonary hypertension (PH), left-heartdisease, HIV, sickle cell anemia, thromboembolisms (CTEPH), sarcoidosis,COPD-associated pulmonary hypertension, pulmonary fibrosis-associatedpulmonary hypertension, chronic obstructive pulmonary disease (COPD),acute respiratory distress syndrome (ARDS), acute lung injury (ALI),alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, pulmonaryemphysema, pulmonary emphysema induced by cigarette smoke, cysticfibrosis (CF), fibrotic disorders, fibrotic disorders of the internalorgans, fibrotic disorders of the lung, fibrotic disorders of the heart,fibrotic disorders of the kidney, fibrotic disorders of the bone marrowfibrotic disorders of the liver, dermatological fibroses, fibrotic eyedisorders, osteodegenerative joint dysfunction, angiotensin-II(AngII)-mediated vasoconstriction, endothelin-1 (ET-1)-mediatedvasoconstriction, ischemic conditions, ischemia associated withmyocardial infarct ischemia associated with wounds, renal pathologies,renal pathologies related to vasoconstriction, hypertension,atherosclerosis, Type 1 diabetes, Type 2 diabetes, coronary arterydisease, scleroderma, stroke, diastolic dysfunction, familialhypercholesterolemia, isolated systolic hypertension, primaryhypertension, secondary hypertension, left ventricular hypertrophy,arterial stiffness associated with long-term tobacco smoking, arterialstiffness associated with obesity, arterial stiffness associated withage, systemic lupus erythematosus, preeclampsia, andhypercholesterolemia.

Disclosed herein is the use of an immunoglobulin fusion protein in themanufacture of a medicament for the treatment of a cardiovasculardisease or condition. The immunoglobulin fusion protein may be any ofthe immunoglobulin fusion proteins disclosed herein. The immunoglobulinfusion protein may comprise an immunoglobulin region attached to one ormore therapeutic peptides. In some embodiments, the therapeutic peptideis attached the amino terminus of the immunoglobulin region. Thecardiovascular disease or condition may be acute heart failure. Thecardiovascular disease or condition may be cardiac hypertrophy. Theimmunoglobulin region may comprise one or more immunoglobulin domains.The immunoglobulin domain may be an immunoglobulin A, an immunoglobulinD, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. Theimmunoglobulin domain may be an immunoglobulin heavy chain region orfragment thereof. The immunoglobulin domain may be an immunoglobulinlight chain region or fragment thereof. The immunoglobulin domain may befrom an anti-viral, anti-bacterial, anti-parasitic, and/or anti-fungalimmunoglobulin. In some instances, the immunoglobulin domain is from amammalian immunoglobulin. Alternatively, the immunoglobulin domain isfrom a chimeric immunoglobulin. The immunoglobulin domain may be from anengineered immunoglobulin or recombinant immunoglobulin. Theimmunoglobulin domain may be from a humanized, human engineered or fullyhuman immunoglobulin. The mammalian immunoglobulin may be a bovineimmunoglobulin. The mammalian immunoglobulin may be a humanimmunoglobulin. In other instances, the mammalian immunoglobulin is amurine immunoglobulin. The immunoglobulin fusion protein, immunoglobulinregion and/or therapeutic peptide may further comprise one or morelinkers. The linker may attach the therapeutic peptide to theimmunoglobulin region. The therapeutic peptide may be a peptide orderivative or variant thereof. Alternatively, therapeutic peptide is asmall molecule. The therapeutic peptide may be relaxin.

Disclosed herein is the use of an immunoglobulin fusion protein in themanufacture of a medicament for the treatment of a cardiovasculardisease or condition, wherein the immunoglobulin fusion proteincomprises a heavy chain fusion having an amino acid sequence at least90% identical to SEQ ID NOs: 20 or 22 and a light chain having an aminoacid sequence at least 90% identical to SEQ ID NOs: 10-12. In someembodiments, the immunoglobulin fusion protein comprises a heavy chainfusion having an amino acid sequence at least 90% identical to SEQ IDNOs: 22 and a light chain having an amino acid sequence at least 90%identical to SEQ ID NOs: 12. The composition may further comprise apharmaceutically acceptable carrier. The subject may be a mammal. Themammal may be a human. The cardiovascular disease may comprise one ormore of acute decompensated heart failure, right heart failure, leftheart failure, global failure, ischemic cardiomyopathy, dilatedcardiomyopathy, heart failure associated with congenital heart defects,heart failure associated with heart valve defects, mitral stenosis,mitral insufficiency, aortic stenosis, aortic insufficiency, tricuspidstenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary valveinsufficiency, heart failure associated with combined heart valvedefects, myocardial inflammation (myocarditis), chronic myocarditis,acute myocarditis, viral myocarditis, diabetic heart failure, alcoholiccardiomyopathy, heart failure associated with cardiac storage disorders,diastolic heart failure, and systolic heart failure.

Further disclosed herein are uses of an immunoglobulin fusion proteinfor the treatment of a disease or condition. Disclosed herein is the useof an immunoglobulin fusion protein for the treatment of a disease orcondition in a subject in need thereof. The immunoglobulin fusionprotein may be any of the immunoglobulin fusion proteins disclosedherein. The immunoglobulin fusion protein may comprise an immunoglobulinregion attached to one or more therapeutic peptides. In someembodiments, the therapeutic peptide is attached the amino terminus ofthe immunoglobulin region. The immunoglobulin region may comprise one ormore immunoglobulin domains. The immunoglobulin domain may be animmunoglobulin A, an immunoglobulin D, an immunoglobulin E, animmunoglobulin G, or an immunoglobulin M. The immunoglobulin domain maybe an immunoglobulin heavy chain region or fragment thereof. Theimmunoglobulin domain may be an immunoglobulin light chain region orfragment thereof. The immunoglobulin domain may be from an anti-viral,anti-bacterial, anti-parasitic, and/or anti-fungal immunoglobulin. Insome instances, the immunoglobulin domain is from a mammalianimmunoglobulin. Alternatively, the immunoglobulin domain is from achimeric immunoglobulin. The immunoglobulin domain may be from anengineered immunoglobulin or recombinant immunoglobulin. Theimmunoglobulin domain may be from a humanized, human engineered or fullyhuman immunoglobulin. The mammalian immunoglobulin may be a bovineimmunoglobulin. The mammalian immunoglobulin may be a humanimmunoglobulin. In other instances, the mammalian immunoglobulin is amurine immunoglobulin. The immunoglobulin fusion protein, immunoglobulinregion and/or therapeutic peptide may further comprise one or morelinkers. The linker may attach therapeutic peptide to the immunoglobulinregion. The therapeutic peptide may be a peptide or derivative orvariant thereof. The therapeutic peptide may be relaxin. The disease orcondition may be an autoimmune disease, heteroimmune disease orcondition, inflammatory disease, pathogenic infection, thromboembolicdisorder, respiratory disease or condition, metabolic disease, centralnervous system (CNS) disorder, bone disease or cancer. In otherinstances, the disease or condition is a blood disorder. In someinstances, the disease or condition is obesity, diabetes, osteoporosis,anemia, or pain. The disease or condition may be a growth disorder.

Disclosed herein is the use of an immunoglobulin fusion protein for thetreatment of a cardiovascular disease or condition in a subject in needthereof. In some embodiments, the immunoglobulin fusion protein treats adisease or condition selected from heart failure, acute coronarysyndrome, atrial fibrillation, cardiac fibrosis, and coronary arterydisease. The immunoglobulin fusion protein may be any of theimmunoglobulin fusion proteins disclosed herein. The immunoglobulinfusion protein may comprise an immunoglobulin region attached to one ormore therapeutic peptides. In some embodiments, the therapeutic peptideis attached the amino terminus of the immunoglobulin region. Thecardiovascular disease or condition may be acute heart failure. Thecardiovascular disease or condition may be cardiac hypertrophy. Theimmunoglobulin region may comprise one or more immunoglobulin domains.The immunoglobulin domain may be an immunoglobulin A, an immunoglobulinD, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. Theimmunoglobulin domain may be an immunoglobulin heavy chain region orfragment thereof. The immunoglobulin domain may be an immunoglobulinlight chain region or fragment thereof. The immunoglobulin domain may befrom an anti-viral, anti-bacterial, anti-parasitic, and/or anti-fungalimmunoglobulin. In some instances, the immunoglobulin domain is from amammalian immunoglobulin. Alternatively, the immunoglobulin domain isfrom a chimeric immunoglobulin. The immunoglobulin domain may be from anengineered immunoglobulin or recombinant immunoglobulin. Theimmunoglobulin domain may be from a humanized, human engineered or fullyhuman immunoglobulin. The mammalian immunoglobulin may be a bovineimmunoglobulin. The mammalian immunoglobulin may be a humanimmunoglobulin. In other instances, the mammalian immunoglobulin is amurine immunoglobulin. The immunoglobulin fusion protein, immunoglobulinregion and/or therapeutic peptide may further comprise one or morelinkers. The linker may attach the therapeutic peptide to theimmunoglobulin region. The therapeutic peptide may be a peptide orderivative or variant thereof. The therapeutic peptide may be relaxin.

Disclosed herein is the use of an immunoglobulin fusion protein for thetreatment of a cardiovascular disease or condition in a subject in needthereof, wherein the immunoglobulin fusion protein comprises a heavychain fusion having an amino acid sequence at least 90% identical to SEQID NOs: 20 or 22 and a light chain having an amino acid sequence atleast 90% identical to SEQ ID NOs: 10-12. In some embodiments, theimmunoglobulin fusion protein comprises a heavy chain fusion having anamino acid sequence at least 90% identical to SEQ ID NOs: 22 and a lightchain having an amino acid sequence at least 90% identical to SEQ IDNOs: 12. The composition may further comprise a pharmaceuticallyacceptable carrier. The subject may be a mammal. The mammal may be ahuman. The cardiovascular disease may comprise one or more of acutedecompensated heart failure, right heart failure, left heart failure,global failure, ischemic cardiomyopathy, dilated cardiomyopathy, heartfailure associated with congenital heart defects, heart failureassociated with heart valve defects, mitral stenosis, mitralinsufficiency, aortic stenosis, aortic insufficiency, tricuspidstenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary valveinsufficiency, heart failure associated with combined heart valvedefects, myocardial inflammation (myocarditis), chronic myocarditis,acute myocarditis, viral myocarditis, diabetic heart failure, alcoholiccardiomyopathy, heart failure associated with cardiac storage disorders,diastolic heart failure, and systolic heart failure.

Pharmacological Properties

Further disclosed herein are methods of improving one or morepharmacological properties of a therapeutic peptide. The method maycomprise producing an immunoglobulin fusion protein disclosed herein.Examples of pharmacological properties may include, but are not limitedto, half-life, stability, solubility, immunogenicity, toxicity,bioavailability, absorption, liberation, distribution, metabolization,and excretion. Liberation may refer to the process of releasing of atherapeutic peptide from the pharmaceutical formulation. Absorption mayrefer to the process of a substance entering the blood circulation.Distribution may refer to the dispersion or dissemination of substancesthroughout the fluids and tissues of the body. Metabolization (orbiotransformation, or inactivation) may refer to the recognition by anorganism that a foreign substance is present and the irreversibletransformation of parent compounds into daughter metabolites. Excretionmay refer to the removal of the substances from the body.

The half-life of a therapeutic peptide may greater than the half-life ofthe non-conjugated therapeutic peptide. The half-life of the therapeuticpeptide may be greater than 4 hours, greater than 6 hours, greater than12 hours, greater than 24 hours, greater than 36 hours, greater than 2days, greater than 3 days, greater than 4 days, greater than 5 days,greater than 6 days, greater than 7 days, greater than 8 days, greaterthan 9 days, greater than 10 days, greater than 11 days, greater than 12days, greater than 13 days, or greater than 14 days when administered toa subject. The half-life of the therapeutic peptide may be greater than4 hours when administered to a subject. The half-life of the therapeuticpeptide may be greater than 6 hours when administered to a subject.

The half-life of the therapeutic peptide may increase by at least about2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 or more hours. The half-life ofthe therapeutic peptide may increase by at least about 2 hours. Thehalf-life of the therapeutic peptide may increase by at least about 4hours. The half-life of the therapeutic peptide may increase by at leastabout 6 hours. The half-life of the therapeutic peptide may increase byat least about 8 hours.

The half-life of a therapeutic peptide may be at least about 1.5, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-foldgreater than the half-life of the non-conjugated therapeutic peptide.The half-life of a therapeutic peptide an immunoglobulin describedherein may be at least about 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,or 50-fold greater than the half-life of the non-conjugated therapeuticpeptide. The half-life of a therapeutic peptide an immunoglobulindescribed herein may be at least about 2-fold greater than the half-lifeof the non-conjugated therapeutic peptide. The half-life of atherapeutic peptide an immunoglobulin described herein may be at leastabout 5-fold greater than the half-life of the non-conjugatedtherapeutic peptide. The half-life of a therapeutic peptide animmunoglobulin described herein may be at least about 10-fold greaterthan the half-life of the non-conjugated therapeutic peptide.

The half-life of a therapeutic peptide an immunoglobulin describedherein may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% greater than thehalf-life of the non-conjugated therapeutic peptide. The half-life of atherapeutic peptide an immunoglobulin described herein may be at leastabout 10% greater than the half-life of the non-conjugated therapeuticpeptide. The half-life of a therapeutic peptide an immunoglobulindescribed herein may be at least about 20% greater than the half-life ofthe non-conjugated therapeutic peptide. The half-life of a therapeuticpeptide an immunoglobulin described herein may be at least about 30%greater than the half-life of the non-conjugated therapeutic peptide.The half-life of a therapeutic peptide an immunoglobulin describedherein may be at least about 40% greater than the half-life of thenon-conjugated therapeutic peptide. The half-life of a therapeuticpeptide an immunoglobulin described herein may be at least about 50%greater than the half-life of the non-conjugated therapeutic peptide.

EXAMPLES

The activity data provided in the following examples are generallyobtained using the immunoglobulin fusion proteins defined in the exampleand exemplified by the provided SEQ ID. It is to be understood that theactivities of any immunoglobulin fusion protein disclosed herein may beenhanced or attenuated depending on conditions not relating toimmunoglobulin fusion protein sequence, for example, expression andpurification conditions.

Example 1: Construction of Palivizumab-Relaxin Fusion Protein Vectorsfor Expression in Mammalian Cells

Relaxin nucleic acid sequences were synthesized by IDT (IA, USA), andamplified by polymerase chain reaction (PCR).

The relaxin2 (9GS) (SEQ ID NO: 33) was genetically fused to nucleicacids encoding for a palivizumab heavy chains 1, 2 or 3 (SEQ ID NOs: 6,7, 8, respectively) using a connecting nucleic acid sequence encodingfor the connecting peptide 16GS (SEQ ID NO: 60) by overlap PCR togenerate palivizumab(NH1, 2, or 3, 16GS)-relaxin2(9GS) (SEQ ID NOs: 18,18, 19, respectively). The pPalivizumab(NH1, 2, or 3,16GS)-relaxin2(9GS) mammalian expression vectors encoding forpalivizumab(NH1, 2, or 3, 16GS)-relaxin2(9GS) were created by in-frameligation of the amplified palivizumab(NH1, 2, or 3, 16GS)-relaxin2(9GS)to the pFuse backbone vector (InvivoGen, CA).

The gene encoding for palivizumab light chain (SEQ ID NO: 1) wasamplified and closed into the pFuse vector to generate a pPalivizumab(L)mammalian expression vector. The resulting mammalian expression vectorswere verified by DNA sequencing.

Example 2: Expression and Purification of Palivizumab-Relaxin FusionProteins

Palivizumab-relaxin heavy chain fusion proteins were each expressedthrough co-transfection of freestyle HEK293 cells withpalivizumab-relaxin heavy chain mammalian expression vectors describedin Example 1 and a palivizumab light chain mammalian expression vector.The cells were grown in shaker flasks at 125 rpm with freestyle 293expression medium (Life Technologies) at 37° C. with 5% CO2. Expressedproteins were secreted into the culture medium and harvested twice every48 hours after transfection. The fusion proteins were purified byProtein A/G chromatography (Thermo Fisher Scientific, IL) and analyzedby SDS-PAGE gel. Purified heavy chain fusion proteins expressed withpalivizumab light chain are shown in the SDS-PAGE gels of FIG. 1A, FIG.1B, and FIG. 1C. For each gel, the first lane corresponds to a molecularmarker, the second lane corresponds to purified protein, and the thirdlane corresponds to purified protein treated with the reducing agentDTT. FIG. 1A shows purified palivizumab(NH1, 16GS)-relaxin2(9GS) (SEQ IDNO: 20) and palivizumab L (SEQ ID NO: 9), collectively, SCX1. FIG. 1Bshows purified palivizumab(NH2, 16GS)-relaxin2(9GS) (SEQ ID NO: 21) andpalivizumab L (SEQ ID NO: 9), collectively, SCX2. FIG. 1C shows purifiedpalivizumab(NH3, 16GS)-relaxin2(9GS) (SEQ ID NO: 22) and palivizumab L(SEQ ID NO: 9), collectively, SCX3.

Example 3: Activity of Palivizumab-Relaxin Fusion Proteins to ActivateRelaxin Receptors

The activities of palivizumab-relaxin fusion proteins purified inExample 2 were examined by a luciferase assay. HEK293 cellsoverexpressed with relaxin receptor (LGR7) and cAMP responsive element(CRE)-luciferase (Luc) reporter gene were grown in DMEM supplementedwith 10% FBS at 37° C. with 5% CO2. Cells were seeded in 384-well platesfor 24 hours and subsequently independently treated with variousconcentrations of palivizumab-relaxin fusion proteins purified fromExample 2 (SCX1, SCX2, or SCX3) or relaxin2 peptide for an additional 24hours. Luminescence intensities were then measured using One-Glo(Promega) luciferase reagent by following manufacturer's instruction.The EC₅₀ values were determined by fitting data into a logisticsigmoidal function: y=A2+(A1−A2)/(1+(x/x0)p), where A1 is the initialvalue, A2 is the final value, x0 is the inflection point of the curve,and p is the power. The EC₅₀ for relaxin-2 was 22.3 pM.

The EC₅₀ for SCX1 was 69.1 pM. The EC₅₀ for SCX2 was 61.2 pM. The EC₅₀for SCX3 was 129 pM. The corresponding cures are shown in FIG. 2A(SCX1), FIG. 2B (SCX2), and FIG. 2C (SCX3).

Example 4: Binding of Palivizumab-Relaxin Fusion Proteins to RSV-Epitope

To test the reduction of palivizumab-relaxin fusion protein binding toRSV-epitope, an RSV binding assay was performed. Briefly, 0.1 ug of RSVF protein was coated in a 96 well plate. Palivizumab-relaxin fusionproteins as described in Example 2, SCX1, SCX2, and SCX3, and a nullpalivizumab antibody were added to separate wells. Anti-human IgG (Fcspecific) at a 1:2,000 dilution in 2% BSA, PBS was added to each well.Fluorescence intensity was measured after 25 and 50 minutes. Plots ofFluorescence intensity vs. fusion protein concentration are shown inFIG. 3A (25 min) and FIG. 3B (50 min). Tables 11 and 12 show thecorresponding data for each construct tested.

TABLE 11 Fusion protein binding assay to RSV-epitope (25 min) ProteinYield LGR7 LGR8 Kd name Heavy chain Light chain (mg/L) (nM) (nM) (nM)SCX1 Palivizumab(NH1, Palivizumab 18.7 0.1285 939.2 111.916GS)-relaxin2(9GS) L SCX2 Palivizumab(NH2, Palivizumab 18 0.1154 326.614.4 16GS)-relaxin2(9GS) L SCX3 Palivizumab(NH3, Palivizumab 18 0.114616290 163.3 16GS)-relaxin2(9GS) L SC3 Palivizumab (null, Palivizumab 250.205 177.1 0.425 16GS)-relaxin2(9GS) L Palivizumab null Palivizumab(null) Palivizumab L 11.6 N/D N/D 0.80

TABLE 12 Fusion protein binding assay to RSV-epitope (50 min) ProteinYield LGR7 LGR8 Kd name Heavy chain Light chain (mg/L) (nM) (nM) (nM)SCX1 Palivizumab(NH1, Palivizumab 18.7 0.1285 939.2 83.116GS)-relaxin2(9GS) L SCX2 Palivizumab(NH2, Palivizumab 18 0.1154 326.69.034 16GS)-relaxin2(9GS) L SCX3 Palivizumab(NH3, Palivizumab 18 0.114616290 107.1 16GS)-relaxin2(9GS) L SC3 Palivizumab (null, Palivizumab 250.205 177.1 0.249 16GS)-relaxin2(9GS) L Palivizumab null Palivizumab(null) Palivizumab L 11.6 N/D N/D 0.49

Example 5: Construction of Optimized Palivizumab-Relaxin Fusion ProteinAntibodies

Optimized palivizumab-relaxin fusion protein antibodies were created bypairing various heavy chain mutations with various light chainmutations. Heavy chain mutations included: NO (SEQ ID NO: 13), N99F,W100G, Y100_(a)G (SEQ ID NO: 22), and W100A (SEQ ID NO: 20). Light chainmutations included germline (SEQ ID NO: 10), S92N (SEQ ID NO: 11), andG91Y (SEQ ID NO: 12). Fc mutations included WT and E233P, L234V, L235A,ΔG236, A327G, A330S, and P331S. Construct components are summarized inTable 13 below.

TABLE 13 Optimized fusion protein summary Heavy chain Light chainMutations Mutations Constructs (CDR3H) (CDR3L) Fc RLX300 NO * E233P,L234V, L235A, (SC3 · s) ΔG236, A327G, A330S, and P331S RLX100 NO * WTRLX303 N99F, W100G, S92N* WT intact Y100_(a)G* RLX301 N99F, W100G, *E233P, L234V, L235A, Y100_(a)G* ΔG236, A327G, A330S, and P331S RLX302N99F, W100G, G91Y* E233P, L234V, L235A, Y100_(a)G* ΔG236, A327G, A330S,and P331S RLX303 N99F, W100G, S92N* E233P, L234V, L235A, Y100_(a)G*ΔG236, A327G, A330S, and P331S RLX304 W100A * E233P, L234V, L235A,ΔG236, A327G, A330S, and P331S RLX305 W100A G91Y* E233P, L234V, L235A,ΔG236, A327G, A330S, and P331S RLX306 W100A S92N* E233P, L234V, L235A,ΔG236, A327G, A330S, and P331S SYN100 NO * WT SYN200 NO * E233P, L234V,L235A, ΔG236, A327G, A330S, and P331S SYN300 N99F, W100G, S92N* E233P,L234V, L235A, Y100_(a)G* ΔG236, A327G, A330S, and P331S

Example 5: Measurement of RSV Binding by Optimized Palivizumab-RelaxinFusion Protein Antibodies

Optimized palivizumab-relaxin fusion protein antibodies were tested forbinding to RSV by ELISA assay. 10 ng/well RSV F protein was coated on96-well plate in PBS 4° C. overnight. The plate was blocked with 2%BSA/PBS at room temperature for 1 hr, then washed twice with 200 μL PBS.The fusion antibodies were incubated at various concentrations (asindicated in FIG. 4) in 2% BSA/PBS at room temperature for 1 hr. Plateswere washed with PBS (0.025% Tween-20 in PBS) four times and washed withPBS one time. Then plates were incubated with horseradishperoxidase-conjugated anti-human IgG (Fc specific) in PBS at roomtemperature for 1 hr, washed with PBS (0.025% Tween-20 in PBS) fivetimes, developed with QuantaBlu fluorogenic peroxidase substrate (ThermoFisher Scientific, IL). Binding was quantified using Spectramaxfluorescence plate reader with excitation at 325 nm and emission at 420nm. Binding curves are shown in FIG. 5. Binding Kd values weredetermined (Table 14).

TABLE 14 Fusion protein binding assay (10 min) Protein Kd (nM) RLX300(no mutations) .301 RLX301 79.3 RLX302 37.7 RLX303 52.1 RLX304 38.2RLX305 75.2 RLX306 82.7 Herceptin 1351

Example 6: Measurement of Thermal Stability of OptimizedPalivizumab-Relaxin Fusion Protein Antibodies

Thermal stability of optimized palivizumab-relaxin fusion proteinantibodies was determined. Results of this assay are shown in Table 15below. The thermal stabilities were measured using a fluorescent proteinbinding dye (Protein Thermal Shift assay, Life Technologies Co., CA).Briefly, Samples with a working volume of 20 μL per well were preparedby mixing 5 ul protein Thermal shift buffer, 2.5 μL of 8× proteinthermal shift Dye with 12.5 μL of antibody (0.5 mg/mL) in DPBS buffer,loaded onto a 96-well 0.2 thin-wall PCR plate, and sealed with SMicroAmp® Optical Adhesive Film. Then a melt curve was run on areal-time PCR instrument. Data were collected at 1° C. intervals from25° C. through 99° C. on the StepOnePlus™ Real-Time PCR System andanalyzed using the Protein Thermal Shift™ Software.

TABLE 15 Fusion protein thermal stability Protein 1^(st) Tm (° C.)2^(nd) Tm (° C.) RLX301 67.83 ± 0.19  85.4 ± 0.76 RLX302 67.95 ± 0.083.25 ± 0.38 RLX303 67.82 ± 0.19 83.66 ± 0.19 RLX304 67.82 ± 0.19 85.53± 0.57 RLX305 68.09 ± 0.19 83.39 ± 0.19 RLX306 68.09 ± 0.19 85.52 ± 0.0

Example 7: Expression and Purification of Optimized Palivizumab-RelaxinFusion Proteins

Palivizumab-relaxin heavy chain fusion proteins were each expressedthrough co-transfection of freestyle HEK293 cells withpalivizumab-relaxin heavy chain mammalian expression vectors describedin Example 5. The cells were grown in shaker flasks at 125 rpm withfreestyle 293 expression medium (Life Technologies) at 37° C. with 5%CO2. Expressed proteins were secreted into the culture medium andharvested twice every 48 hours after transfection. The fusion proteinswere purified by Protein A/G chromatography (Thermo Fisher Scientific,IL) and analyzed by SDS-PAGE gel. Purified heavy chain fusion proteinsexpressed with palivizumab light chain are shown in the SDS-PAGE gels ofFIGS. 5A-F. For each gel, the first lane corresponds to a molecularmarker, the second lane corresponds to purified protein, and the thirdlane corresponds to purified protein treated with the reducing agentDTT. FIG. 5A shows purified RLX301 (SEQ ID NO: 22) and (SEQ ID NO: 10).FIG. 5B shows purified RLX302 (SEQ ID NO: 22) and (SEQ ID NO: 12). FIG.5C shows purified RLX303 (SEQ ID NO: 22) and (SEQ ID NO: 11). FIG. 5Dshows purified RLX304 (SEQ ID NO: 20) and (SEQ ID NO: 10). FIG. 5E showspurified RLX305 (SEQ ID NO: 20) and (SEQ ID NO: 12). FIG. 5F showspurified RLX306 (SEQ ID NO: 20) and (SEQ ID NO: 11).

Example 8: Activity of Optimized Palivizumab-Relaxin Fusion Proteins toActivate Relaxin Receptors

The activities of optimized palivizumab-relaxin fusion proteins purifiedin Example 7 were examined by a luciferase assay. HEK293 cellsoverexpressed with relaxin receptor (LGR7) and cAMP responsive element(CRE)-luciferase (Luc) reporter gene were grown in DMEM supplementedwith 10% FBS at 37° C. with 5% CO2. Cells were seeded in 384-well platesfor 24 hours and subsequently independently treated with variousconcentrations of palivizumab-relaxin fusion proteins purified fromExample 7 (RLX301, RLX302, RLX303, RLX304, RLX305, or RLX306) orrelaxin2 peptide for an additional 24 hours. Luminescence intensitieswere then measured using One-Glo (Promega) luciferase reagent byfollowing manufacturer's instruction.

The EC₅₀ values were determined by fitting data into a logisticsigmoidal function: y=A2+(A1−A2)/(1+(x/x0)p), where A1 is the initialvalue, A2 is the final value, x0 is the inflection point of the curve,and p is the power. The EC₅₀ for relaxin-2 was 22 pM. The EC₅₀ forRLX301 was 72 pM. The EC₅₀ for RLX302 was 179 pM. The EC₅₀ for RLX303was 86 pM. The EC₅₀ for RLX304 was 78 pM. The EC₅₀ for RLX305 was 98 pM.The EC₅₀ for RLX306 was 153 pM. The corresponding cures are shown inFIG. 6A (RLX301), FIG. 6B (RLX302), FIG. 6C (RLX303), FIG. 6D (RLX304),FIG. 6E (RLX305), and FIG. 6F (RLX306).

Activities of optimized palivizumab-relaxin fusion proteins, RLX 302 andRLX303, were further examined using UL-hLGR7-Cre-Luc reporter cell line.The EC₅₀ for relaxin-2 was 20 pM and SA was 1000 units/μg. The EC₅₀ forRLX302 was 130 pM and SA was 5.9 units/μg. The EC₅₀ for RLX303 was 93 pMand SA was 8.0 units/μg. The corresponding curves are shown in FIG. 7A(RLX302) and FIG. 7B (RLX303).

Example 9: RLX303 Protein Characterization

RLX303 was characterized by analytical size-excusion chromatography andliquid chromatography-mass spectroscopy. RLX303 was confirmed to contain96% of RLX303 monomer protein with correct light chain and heavy chainmasses. Protein concentration was found to be 26-29 mg/ml in PBS (pH7.4). Analytical size-exchange chromatography is shown in FIG. 8A.Liquid chromatography-mass spectroscopy is shown in FIG. 8B.

Example 10: Relaxin-Ig Fusion Serum Stability

Serum stability of Relaxin-2 peptide, RLX302, and RLX303 was determined.Proteins were incubated with serum, then activity was measured incell-based assays. Relaxin-Ig fusions remained stable over 5 days inhuman serum with about 15% loss of activity in cyno serum. Over 95% ofhuman relaxin-2 peptide is gone after 5 days with degradation more rapidin cyno serum than in human serum. Sample concentrations were determinedby ELISA and LC/MS was used to evaluate the chemical stability of theRelaxin-Ig fusions in serum. Stability curves are shown in FIG. 9A(Relaxin-2 peptide), FIG. 9B (RLX302), and FIG. 9C (RLX303).

Example 11: Relaxin Ig-Fusion PK Analysis

Pharmacokinetic properties of RLX302 and RLX303 were evaluated in miceusing a single IV injection in CD1 mice at a dose of 3.7 mg/kg (n=3).Comparable exposure and half-life was found between the RLX302 andRLX303 fusion proteins. Pharmacokinetic data for the mouse experimentsis found in Table 16. Pharmacokinetic curves for the mouse experimentsare shown in FIG. 10.

TABLE 16 Mouse PK Analysis Protein T_(1/2) (hr) T_(max) (hr) C_(max)(nmol/L) AUC (nmol · hr/L) RLX302 63.78 0.5 137.06 3023.74 RLX303 68.180.5 178.29 2372.33

Pharmacokinetic properties of RLX302 and RLX303 were evaluated through asingle i.v. or s.c injection in SD1 rats at a dosage of 2.1 mg/kg (n=3).Comparable exposure and half-life was found between the RLX302 andRLX303 fusion proteins. For s.c. dosing, in the RLX302 dosed animals,samples from 2 out 3 rats at timepoint 336 hr were out of detectionrange. In the RLX303 s.c. dosed animals, all animals were below assaydetection at timepoint 336 hr Pharmacokinetic data for the ratexperiments is found in Table 17. Pharmacokinetic curves for the ratexperiments are shown in FIG. 11A and FIG. 11B.

TABLE 17 Rat PK Analysis AUC Dosing Route Protein T_(1/2) (hr) T_(max)(hr) C_(max) (nmol/L) (nmol · hr/L) I.V. RLX302 68 0.33 226 1756 RLX30385 0.33 176 1750 S.C. RLX302 91 48 11 1780 RLX303 91 48 17 2795

Example 12: Mouse Pharmacodynamics Analysis

Pharmacodynamic properties of RLX302 and RLX303 were evaluated using as.c. dose between 0.02 to 0.6 mg/kg in estrogen-primed CD1 female mice(n=8-12). Mice were treated with human relaxin-2 peptide as a controlInterpubic ligament width was measured at 1 week post dosing.Pharmacodynamic data is shown in FIG. 12A. Mice were treated with humanrelaxin-2 peptide as a control and pubic symphasis width was measured 24hours post injection. Control data is shown in FIG. 12B.

Example 13: Phase 1 Clinical Trials

Cohorts of 10 volunteers, 8 ‘active’ and 2 placebo, are recruited forsingle ascending dose safety evaluation and PD measures of RLX302 andRLX303 in healthy volunteers. Five dose-levels are used having astarting dose ˜0.1 mg/kg IV, half-log escalating dose groups (0.1, 0.3,1.0, 3.0, 10 mg/kg). Intravenous doses are used initially to supportfuture acute/subacute indications where rapid onset of action ispreferred (based on differences seen preclinically); also allowsdiscontinuation of infusion if acute adverse events occur. 2-3dose-levels are used (high, mid, +/−low) with subcutaneous (SC) dosingto establish SC safety, local tolerability, PK, and bioavailability.Additional subjects (optional) are specified in the protocol to add upto 2 lower or intermediate dose groups depending onsafety/tolerability/PD effects. Patients are followed for 3 months forPK, safety.

For multiple ascending dose safety evaluation, 5 dose groups are used:1, 3, 10 mg/kg SC Q2w and 1.5 mg/kg Q1w dosing. Q2w group receives 7 SCinjections over 12 weeks; Q1w group receives 13 injections over 12weeks. Cohorts of ten healthy volunteers, 8 ‘active’ and 2 placebo,safety evaluation and PD measures in healthy volunteers. The goal is toestablish safety/tolerability/PK and PD across dose range in healthyvolunteers. Potential for immunogenicity (ADAs) is evaluated usingpooled data. Additional subjects (optional 2 cohorts) specified in theprotocol to explore lower, intermediate, and alternate frequency (Qmonth, additional Q1w groups) Total follow up for 5 months (from initialdose) for PK, safety.

Example 14: Phase 1b Clinical Trials

Long-acting relaxin-immunoglobulin fusion, RLX302 is studied in a Phase1b clinical trial for reducing heart failure hospitalization,cardiovascular death, and dyspnea. The primary objective is, usingparallel groups, randomized, Pbo-controlled safety, tolerability, PK andPD study in subjects with compensated stable HFrEF to support the safetyassessment and dose range selection for a chronic dosing Phase 2 Programin chronic heart failure by: 1) Characterizing the safety andtolerability of subcutaneous RLX302 in patients with stable compensatedHFrEF and 2) Characterize the PK of subcutaneous RLX302 in patients withHFrEF. The secondary objective is: 1) characterize the cardiac PDeffects of RLX302 using blood and imaging markers in a population ofHFrEF patients with baseline elevated NT-proBNP (>600 pg/ml—TBD) and 2)Characterize the renal PD effects of RLX302 in a population of HFrEFpatients with baseline elevated NT-proBNP (>600 pg/ml—TBD). Tertiaryobjectives include characterizing the effects of RLX302 on markers ofcardiac damage and clinical outcomes related to RLX302 effect on cardiacand renal physiology. Primary endpoints are: 1) Safety measurementsincluding standard clinical and laboratory evaluations (BP, din labs,ECG, etc.), 2) PK, and 3) immunogenicity assessments (ADAs, AEs).Secondary endpoints include: 1) Cardiac biomarkers: NT-proBNP, 2)Cardiac Imaging (transthoracic echocardiography): Left Atrial size, EF,strain, diastolic function, remodeling indices, and 3) Renalphysiology/injury markers: renal blood flow, GFR, cystatin C, KIM1,plasma BUN & creatinine. Tertiary endpoints include: 1) MACE events, HFhospitalization, CV death, all cause mortality; 2) composite clinicalassessment (NYHA classification, Pt Global Assessment, MACE); 3)Hospitalization for renal failure; and 4) Adjustments in diuretics andother HF medications (evidence of intensification of therapy orpotential diuretic sparing effect) from baseline. The strategic goalsare: 1) Early capture of potential mechanism-based biologic and clinicalbenefits to enable accelerated investment in further developmentactivities (chronic tox, Ph 2 startup, etc.) and 2) Trigger transitionpoints in collaboration. Study rationale is to assess the safety,tolerability, and early signs of efficacy for RLX302 in the majoreventual target population of HFrEF patients. Strong biologic and/orclinical signals of treatment benefit would enhance confidence forfurther investment and potential timeline acceleration. Key biomarkerobjectives include ≥30% reduction in NT-proBNP during first week oftreatment. Key inclusion criteria are: HFrEF, NYHA class Recent HFhospitalization within 3-6 months, other enrichment criteria for highrisk HF e.g. NT-proBNP≥600-900 pg/mL, and eGFR (limit to be determinedbased on projected impact on enrollment). Key exclusion criteria areHFpEF, CrCl<30 (advanced renal dysfunction), and BP restrictions similarto Pre-RELAX AHF/RELAX AHF/PARADIGM (e.g. SBP<100). Key features of thetrial are: 1) randomized, double blind, multiple dose; 2) HFrEFpatients, 3) four week exposure (2 SC doses), 4 weeks timepoint frombaseline=primary endpoint; assessments continued at weeks 8 and 12(study completion); 4) 2-3 dose levels+placebo (approx. 120 subjectstotal); and 5) interim analysis based on 1 or 2 wk follow up data and75% recruitment—potential to trigger investment in chronic tox and/ormanufacturing of drug product. Treatment arms will include PBO, doselevels MTD, mid- and low-dose guided by SAD in HV. Sample sizes will be30-40 subjects per arm as guided by available data.

Clinically meaningful, dose dependent changes in multiple aspects ofpotential therapeutic benefit are observed (as outlined in the keybiomarker objectives and below). Signals are concordant and ofsufficient magnitude to reasonably preclude a chance finding.Therapeutic benefits include: reduction in NT-proBNP, reduction inhs-TnI/T, improvement in Echo parameters, e.g., reduction in LA size (inpatients with increased LA size at baseline), reduction of clinicalsigns of congestion (clinical signs/diuretic use, LFTs), decreased serumcreatinine and BUN, improved renal hemodynamics (improved RPF withstable or improved GFR ±reduced Filtration Fraction), improvement ofdyspnea (symptom scores; includes DOE, PND), worsening HF over 4-12weeks (during active treatment phase and/or follow-up period), hospitaladmission for AHF or Renal Failure over 4 weeks (during active treatmentphase and/or follow-up period). Treatment with RLX302 results inimproved outcomes with more convenient dosing schedules than currentrelaxin-based treatments (e.g. serelaxin).

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof.

Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of the present invention isembodied by the appended claims.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

All references cited herein are incorporated by reference in theirentirety and for all purposes to the same extent as if each individualpublication or patent or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

TABLE 1 Immunoglobulin Light Chain (LC) and Heavy Chain (HC)-Nucleotide SequenceNAME SEQ ID NO SEQUENCE Palivizumab L 1GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCCGTGGGCGACCGCGTGACCATCACCTGCAAGTGCCAGCTGTCCGTGGGCTACATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCCTCCCGCTTCTCCGGCTCCGGCTCCGGCACCGAGTTCACCCTGACCATCTCCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGCTTCCAGGGCTCCGGCTACCCCTTCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGTCCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGTPalivizumab  L (germline) 2GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCCGTGGGCGACCGCGTGACCATCACCTGCAAGTGCCAGCTGTCCGTGGGCTACATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCCTCCCGCTTCTCCGGCTCCGGCTCCGGCACCGCCTTCACCCTGACCATCTCCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGCTTCCAGGGCTCCGGCTACCCCTTCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGTCCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT Palivizumab L (G91Y) 3GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCCGTGGGCGACCGCGTGACCATCACCTGCAAGTGCCAGCTGTCCGTGGGCTACATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCCTCCCGCTTCTCCGGCTCCGGCTCCGGCACCGCCTTCACCCTGACCATCTCCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGCTTCCAGTACTCCGGCTACCCCTTCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGTCCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT Palivizumab L (S92N) 4GACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCCGTGGGCGACCGCGTGACCATCACCTGCAAGTGCCAGCTGTCCGTGGGCTACATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCCTCCCGCTTCTCCGGCTCCGGCTCCGGCACCGCCTTCACCCTGACCATCTCCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGCTTCCAGGGCAACGGCTACCCCTTCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGTCCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT Palivizumab H 5CAGGTGACCCTGCGCGAGTCCGGCCCTGCACTGGTGAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCTCCCTGTCCACCTCCGGCATGTCCGTGGGCTGGATCCGGCAGCCTCCCGGCAAGGCCCTGGAGTGGCTGGCTGACATCTGGTGGGACGACAAGAAGGACTACAACCCCTCCCTGAAGTCCCGCCTGACCATCTCCAAGGACACCTCCAAGAACCAGGTGGTGCTGAAGGTGACCAACATGGACCCCGCCGACACCGCCACCTACTACTGCGCCCGCTCAATGATTACCAACTGGTACTTCGACGTGTGGGGAGCCGGTACCACCGTGACCGTGTCTTCCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAACCCAAATCTTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCAGTCGCCGGACCGTCAGTCTTCCTCTTCCCTCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAAGCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCTCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATAA Palivizumab H2 6CAGGTGACCCTGCGCGAGTCCGGCCCTGCACTGGTGAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCTCCCTGTCCACCTCCGGCATGTCCGTGGGCTGGATCCGGCAGCCTCCCGGCAAGGCCCTGGAGTGGCTGGCTGACATCTGGTGGGACGACAAGAAGGACTACAACCCCTCCCTGAAGTCCCGCCTGACCATCTCCAAGGACACCTCCAAGAACCAGGTGGTGCTGAAGGTGACCAACATGGACCCCGCCGACACCGCCACCTACTACTGCGCCCGCTCAATGATTACCAACGCCTACTTCGACGTGTGGGGAGCCGGTACCACCGTGACCGTGTCTTCCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAACCCAAATCTTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCAGTCGCCGGACCGTCAGTCTTCCTCTTCCCTCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAAGCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCTCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATAA Palivizumab H3 7

Palivizumab H4  8

TABLE 2 Immunoglobulin Light Chain (LC) and Heavy Chain (HC)-Amino Acid SequenceName SEQ ID NO Sequence Palivizumab L  9DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECPalivizumab L 10 DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLI(germline) YDTSKLASGVPSRFSGSGSGTAFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECPalivizumab L 11 DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLI (G91Y)YDTSKLASGVPSRFSGSGSGTAFTLTISSLQPDDFATYYCFQYSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECPalivizumab L 12 DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLI (S92N)YDTSKLASGVPSRFSGSGSGTAFTLTISSLQPDDFATYYCFQGNGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECPalivizumab H 13 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Palivizumab H1 14QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNAYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Palivizumab H2 15

Palivizumab H3 16

TABLE 3  Immunoglobulin fusion protein-Nucleotide Sequence NAMESEQ ID NO SEQUENCE Palivizumab (NH1,  16GS) Relaxin2 (9GS) 17

Palivizumab (NH2,  16GS) Relaxin2 (9GS) 18

Palivizumab (NH3,  16GS) Relaxin2 (9GS) 19

For SEQ ID NOs: 17-19 Immunoglobulin Region = dashed underlinePeptide/Therapeutic peptide = italic Peptide/Therapeutic peptideinternal linker = italic Connecting peptide = bold, thick underlineLinker = double underline Protease site: underline

TABLE 4  Immunoglobulin fusion protein-Amino Acid Sequence NameSEQ ID NO  Sequence Palivizumab (NH1, 16GS) Relaxin2  (9GS) 20

Palivizumab (NH2, 16GS) Relaxin2  (9GS) 21

Palivizumab (NH3, 16GS) Relaxin2  (9GS) 22

For SEQ ID NOs: 20-22 Immunoglobulin Region = dashed underlinePeptide/Therapeutic peptide = italic Peptide/Therapeutic peptideinternal linker = italic Connecting peptide = bold, thick underlineLinker = double underline Protease site: underline

TABLE 5  Therapeutic Peptides-Nucleotide Sequence NAME SEQ ID NOSEQUENCE Relaxin2 23 GACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCGTGAACTGGTTCGTGCTCAGATCGCTATCTGCGGTATGTCTACCTGGTCTAAACGTTCTCTGTCTCAGGAAGACGCTCCGCAGACCCCGCGTCCGGTTGCTGAAATCGTTCCGTCTTTCATCAACAAAGACACCGAAACCATCAACATGATGTCTGAATTCGTTGCTAACCTGCCGCAGGAACTGAAACTGACCCTGTCTGAAATGCAGCCGGCTCTGCCGCAGCTGCAGCAGCACGTTCCGGTTCTGAAAGACTCTTCTCTGCTGTTCGAAGAATTCAAAAAACTGATCCGTAACCGTCAGTCTGAAGCTGCTGACTCTTCTCCGTCTGAACTGAAATACCTGGGTCTGGACACCCACTCTCGTAAAAAACGTCAGCTGTACTCTGCTCTGGCTAACAAATGCTGCCACGTTGGTTGCACCA AACGTTCTCTGGCTCGTTTCTGCRelaxin2 (XT100) 24

Relaxin2  (XT35)  25

Relaxin2 (single) 26

Relaxin2 (insulin C peptide) 27

Relaxin2  (XT21) 28

Relaxin2a 29 GACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCGTGAACTGGTTCGTGCTCAGATCGCTATCTGCGGTATGTCTACCTGGTCTAAACG T Relaxin2b 30CGTAAAAAACGTCAGCTGTACTCTGCTCTGGCTAACAAATGCTGCCACGTTGGTTGCACCAAACGTTCTCTGGCTCGTTTCTGC Relaxin2  (30GS) 31

Relaxin2  Q60A  (30GS) 32

Relaxin2  (9GS) 33

Relaxin2c  (9GS) 34

Relaxin2  (GGGPRR) 35

Relaxin2  (18GS) 36

For SEQ ID NOs: 23-36 Immunoglobulin Region = dashed underlinePeptide/Therapeutic peptide = italic Peptide/Therapeutic peptideinternal linker = italic Connecting peptide = bold, thick underlineLinker = double underline Protease site: underline

TABLE 6  Therapeutic Peptides-Amino Acid Sequence Name SEQ ID NO Sequence Relaxin2 37 DSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQLYSALA NKCCHVGCTKRSLARFCRelaxin2  (XT100) 38

Relaxin2  (XT35) 39

Relaxin2  (single) 40

Relaxin2 (insulin C 41

peptide) Relaxin2  (XT21) 42

Relaxin2a 43 DSWMEEVIKLCGRELVRAQIAICGMSTWSKR Relaxin2b 44RKKRQLYSALANKCCHVGCTKRSLARFC relaxin A chain 45 QLYSALANKCCHVGCTKRSLARFCrelaxin B chain 46 DSWMEEVIKLCGRELVRAQIAICGMSTWS Relaxin2  (30GS) 47

Relaxin2 Q60A (30GS) 48

Relaxin2 (9GS) 49

Relaxin2c (9GS) 50

Relaxin2 (GGGPRR) 51

Relaxin2 (18GS) 52

For SEQ ID NOs: 37-52 Immunoglobulin Region = dashed underlinePeptide/Therapeutic peptide = italic Peptide/Therapeutic peptideinternal linker = italic Connecting peptide = bold, thick underlineLinker = double underline Protease site: underline

TABLE 7 Connecting Peptide Sequences Name SEQ ID NO Sequence (GGGGS)_(n)53 GGGGS n = 1-10 (GGGGG)_(n) 54 GGGGG n = 1-10 CEXa 55NGGPSSGAPPPSGGGGG CEXb 56 GGPSSGAPPPSGGGGG EAAAK 57 EAAAKEAAAKEAAAKCEXGGGGS 58 GGPSSGAPPPSGGGGS XT21 59 SGSETPGTSESATPESGPGSP 16GS 60GGGGSGGGGSGGGGSG

TABLE 8 Linker Sequences Name SEQ ID NO Sequence Linker a 61 GGGGGLinker b 62 GGGGS

TABLE 9  Internal Linker Sequences Name SEQ ID NO Sequence XT100 63

XT35 64

Insulin C peptide 65

XT21 66

XT35 (noHIS) 67

30GS 68

9GS 69

18GS 70

GGGPRR 71

TABLE 10 Immunoglobulin Amino Acid Sequences SEQ ID NAME NO SEQUENCEHC CDR1 72 TFSGFSLSTSGMSVG HC CDR2 73 DIWWDDKKDYNPSLKS HC CDR3 74SMITNWYFDV original HC CDR3 75 SMITX(1)X(2)X(3)FDV; X(1) is F,A, G, or P; X(2) is G, A, S, T, or P; X(3) is G, A, V, L, or P HC CDR376 SMITX(1)X(2)X(3)FDV; X(1) is F, A, G, or P; X(2) is W, G, A, S,T, or P; X(3) is Y, G, A, V, L, or P HC CDR3 77SMITX(1)X(2)X(3)FDV; X(1) is N, F, A, G, or P; X(2) is G, A, S,T, or P; X(3) is Y, G, A, V, L, or P HC CDR3 78SMITX(1)X(2)X(3)FDV; X(1) is N, F, A, G, or P; X(2) is W, G, A,S, T, or P; X(3) is G, A, V, L, or P HC CDR3 79 SMITFGGFDV HC CDR3 80SMITNAYFDV HC CDR3 81 SMITNGYFDV HC CDR3 82 SMITFWYFDV HC CDR3 83SMITFGYFDV HC CDR3 84 SMITFAYFDV HC CDR3 85 SMITFAGFDV HC CDR3 86SMITNGGFDV HC CDR3 87 SMITNAGFDV HC CDR3 88 SMITNWGFDV LC CDR1 89KCQLSVGYMH LC CDR2 90 DTSKLAS LC CDR3 91 FQGSGYPFT (original) LC CDR3 92FQX(4)X(5)GYPFT; X(4) is G, Y, F, W, P, L, V, or A; X(5) is S,N, G, A, V, L, or P LC CDR3 93 FQX(4)X(5)GYPFT; X(4) is Y, F,W, P, L, V, or A; X(5) is S, N, G, A, V, L, or P LC CDR3 94FQX(4)X(5)GYPFT; X(4) is G, Y, F, W, P, L, V, or A; X(5) is N,G, A, V, L, or P LC CDR3 95 FQYSGYPFT LC CDR3 96 FQGNGYPFT LC CDR3 97FQYNGYPFT HC F_(C) 98 DKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1.-47. (canceled)
 48. An antibody comprising a light chain comprisinglight chain complementarity determining regions CDRL1, CDRL2, and CDRL3,and a heavy chain comprising heavy chain complementarity determiningregions CDRH1, CDRH2, and CDRH3, wherein: the CDRL1 comprises the aminoacid sequence of SEQ ID NO: 89 (KCQLSVGYMH), the CDRL2 comprises theamino acid sequence of SEQ ID NO: 90 (DTSKLAS), the CDRL3 comprises theamino acid sequence of SEQ ID NO: 96 (FQGNGYPFT), the CDRH1 comprisesthe amino acid sequence of SEQ ID NO: 72 (TFSGFSLSTSGMSVG), the CDRH2comprises the amino acid sequence of SEQ ID NO: 73 (DIWWDDKKDYNPSLKS),and the CDRH3 comprises the amino acid sequence of SEQ ID NO: 75(SMITFGGFDV).
 49. The antibody of claim 48, wherein the light chaincomprises a sequence at least 90% identical to SEQ ID NO:
 12. 50. Theantibody of claim 48, wherein the heavy chain comprises a sequence atleast 90% identical to SEQ ID NO:
 16. 51. The antibody of claim 48,wherein the light chain comprises a sequence at least 90% identical toSEQ ID NO: 12, and the heavy chain comprises a sequence at least 90%identical to SEQ ID NO:
 16. 52. The antibody of claim 48, wherein thelight chain comprises SEQ ID NO: 12, and the heavy chain comprises SEQID NO:
 16. 53. A composition comprising a relaxin polypeptide connectedto the antibody of claim
 48. 54. The composition of claim 53, whereinthe relaxin polypeptide is connected to the amino-terminus of theantibody.
 55. The composition of claim 53, wherein the relaxinpolypeptide comprises a B chain and an A chain.
 56. The composition ofclaim 55, wherein the B chain comprises SEQ ID NO:
 46. 57. Thecomposition of claim 55, wherein the A chain comprises SEQ ID NO: 45.58. The composition of claim 55, wherein the B chain comprises SEQ IDNO: 46, and the A chain comprises SEQ ID NO:
 45. 59. The composition ofclaim 53, wherein the relaxin polypeptide comprises a sequence at least90% identical to SEQ ID NO:
 49. 60. The composition of claim 53, whereinthe heavy chain sequence comprises SEQ ID NO:
 22. 61. A compositioncomprising a first polypeptide comprising a relaxin polypeptideconnected to the amino-terminus of a heavy chain antibody variabledomain, and a second polypeptide comprising a light chain antibodyvariable domain.
 62. The composition of claim 61, wherein the firstpolypeptide comprises at least 90% identity to SEQ ID NO: 22 and thesecond polypeptide comprises at least 90% identity to SEQ ID NO:
 12. 63.The composition of claim 62, wherein the relaxin polypeptide comprises aB chain comprising SEQ ID NO: 46, and an A chain comprising SEQ ID NO:45.
 64. The composition of claim 62, wherein the relaxin polypeptidecomprises a sequence at least 90% identical to SEQ ID NO:
 49. 65. Thecomposition of claim 62, wherein the first polypeptide comprises SEQ IDNO: 22 and the second polypeptide comprises SEQ ID NO:
 12. 66. A methodof treating fibrosis in a subject in need thereof, the method comprisingadministering to the subject the antibody of claim
 48. 67. A method oftreating fibrosis in a subject in need thereof, the method comprisingadministering to the subject the composition of claim 61.